Scattered Branched-Chain Fatty Acids And Biological Production Thereof

ABSTRACT

Methods and cells for producing scattered branched-chain fatty acids are provided. For example, the invention provides a method for producing branched-chain fatty acid comprising a methyl on one or more even number carbons. The method comprises culturing a cell comprising an exogenous or overexpressed polynucleotide comprising a nucleic acid sequence encoding a polypeptide that catalyzes the conversion of propionyl-CoA to methylmalonyl-CoA and/or an exogenous or overexpressed polynucleotide comprising a nucleic acid sequence encoding a polypeptide that catalyzes the conversion of succinyl-CoA to methylmalonyl-CoA, under conditions allowing expression of the polynucleotide(s) and production of branched-chain fatty acid. The cell produces more branched-chain fatty acid comprising a methyl on one or more even number carbons than an otherwise similar cell that does not comprise the polynucleotide(s). A cell that produces branched-chain fatty acid and the branched-chain fatty acid also are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/294,274, filed Jan. 12, 2010, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The invention relates to cells and methods for producing fatty acids,and more particularly relates to cells and methods for producingscattered branched-chain fatty acids.

BACKGROUND OF THE INVENTION

Branched-chain fatty acids are carboxylic acids with a methyl or ethylbranch on one or more carbons that can be either chemically synthesizedor isolated from certain animals and bacteria. While certain bacteria,such as Escherichia coli, do not naturally produce branched-chain fattyacids, some bacteria, such as members of the genera Bacillus andStreptomyces, can naturally produce these fatty acids. For example,Streptomyces avermitilis and Bacillus subtilis both producebranched-chain fatty acids with from 14 to 17 total carbons, with thebranches in the iso and anteiso positions (Cropp et al., Can. J.Microbiology 46: 506-14 (2000); De Mendoza et al., Biosynthesis andFunction of Membrane Lipids, in Bacillus subtilis and OtherGram-Positive Bacteria, Sonenshein and Losick, eds., American Societyfor Microbiology (1993)). However, these organisms do not producebranched-chain fatty acids in amounts that are commercially useful.Another limitation of these natural organisms is that they apparently donot produce medium-chain branched-chain fatty acids, such as those with11 or 13 carbons. In addition, if fatty acids having particular chainlengths, branches on particular carbons, or branches at positions otherthan the iso and anteiso positions are desired, these fatty acids maynot be available or easily isolated from a natural organism inmeaningful quantities.

As such, there remains a need for commercially useful,bacterially-produced, branched-chain fatty acids. In addition, thereremains a need for a method of producing such branched-chain fattyacids.

SUMMARY OF THE INVENTION

Methods and cells for producing scattered branched-chain fatty acids areprovided. In certain embodiments, the method for producingbranched-chain fatty acids in a cell includes expressing in the cell oneor more recombinant polypeptides that catalyze the conversion ofmethylmalonyl-CoA to methylmalonyl-ACP; and culturing the cell underconditions suitable for producing the polypeptide, such thatbranched-chain fatty acids are produced.

Also provided is a method for producing branched-chain fatty acids in acell, the method including expressing in the cell one or morerecombinant polypeptides that increase the production ofmethylmalonyl-CoA in the cell; and culturing the cell under conditionssuitable for producing the recombinant polypeptide, such thatbranched-chain fatty acids are produced.

In certain embodiments, a method for producing branched-chain fattyacids in a cell is provided, the method including expressing in the cella polypeptide that has propionyl-CoA synthetase activity; inhibitingpropionylation of the propionyl-CoA synthetase; and culturing the cellunder conditions suitable for producing the polypeptide, such thatbranched-chain fatty acids are produced.

Further provided is a method for producing branched-chain fatty acids ina cell, the method including expressing in the cell a polypeptide thathas methylmalonyl-CoA mutase activity; expressing in a cell apolypeptide that has methylmalonyl-CoA epimerase activity; and culturingthe cell under conditions suitable for producing the polypeptides, suchthat branched-chain fatty acids are produced.

A composition comprising a mixture of biologically-producedbranched-chain fatty acids is also provided. The composition can includebranched-chain fatty acids having a chain length of C12 to C16 and fromabout 1 to about 3 methyl branches positioned on one or moreeven-numbered carbons.

In certain embodiments, a method for producing branched-chain fattyacids in a cell is provided, the method including expressing in the cellone or more recombinant polypeptides that increase the production ofmethylmalonyl-CoA in the cell; expressing in the cell a recombinantpolypeptide that catalyzes the conversion of methylmalonyl-CoA tomethylmalonyl-ACP; and culturing the cell under conditions suitable forproducing the recombinant polypeptide, such that branched-chain fattyacids are produced.

In addition, in certain embodiments, a method for producingbranched-chain fatty acids in a cell is provided, the method includingexpressing in the cell one or more recombinant polypeptides thatincrease the production of methylmalonyl-CoA in the cell; expressing inthe cell a recombinant polypeptide that catalyzes the conversion ofmethylmalonyl-CoA to methylmalonyl-ACP; expressing in the cell arecombinant thioesterase; and culturing the cell under conditionssuitable for producing the recombinant polypeptide, such thatbranched-chain fatty acids are produced.

Also provided is a method for producing branched-chain fatty acids in acell, the branched-chain fatty acids having a chain length from about 10to 18 carbons and branching at the second carbon. The method includesmodifying the cell to increase carbon flow to methylmalonyl-CoA; andculturing the cell under conditions suitable for carbon flow tomethylmalonyl-CoA to be increased, such that branched-chain fatty acidshaving a chain length from about 10 to about 18 carbons and branching atthe second carbon are produced. In certain embodiments, the branchingcan be on the fourth, sixth, eighth, tenth, or twelfth carbon.

In certain embodiments, a method for producing branched-chain fattyacids in a cell is provided, the branched-chain fatty acids having achain length from about 10 to 18 carbons and branching at the secondcarbon. The method includes modifying the cell to generatemethylmalonyl-ACP from methylmalonyl-CoA; and culturing the cell underconditions suitable for generation of methylmalonyl-ACP frommethylmalonyl-CoA, such that branched-chain fatty acids having a chainlength from about 10 to about 18 carbons and branching at the secondcarbon are produced. In certain embodiments, the branching can be on thefourth, sixth, eighth, tenth, or twelfth carbon.

A method for producing modified fatty acids in a cell is also provided,the method including providing a cell having type II fatty acid synthaseactivity; expressing in the cell one or more recombinant polypeptidesthat catalyze formation of at least one intermediate metabolite, whereinthe at least one intermediate metabolite is incorporated by the type IIfatty acid synthase; and culturing the cell under conditions suitablefor producing the recombinant polypeptide, such that modified fattyacids are produced.

Further provided is an Escherichia cell that produces branched-chainfatty acids having a chain length from about 10 to about 18 carbons andcomprising one or more methyl branches on one or more even-numberedcarbons.

The invention further provides a method for producing branched-chainfatty acid comprising a methyl on one or more even number carbons. Themethod comprises culturing a cell comprising (aa) an exogenous oroverexpressed polynucleotide comprising a nucleic acid sequence encodinga polypeptide that catalyzes the conversion of propionyl-CoA tomethylmalonyl-CoA and/or (bb) an exogenous or overexpressedpolynucleotide comprising a nucleic acid sequence encoding a polypeptidethat catalyzes the conversion of succinyl-CoA to methylmalonyl-CoA. Thecell is cultured under conditions allowing expression of thepolynucleotide(s) and production of branched-chain fatty acid.Optionally, the method further comprises extracting from the culture thebranched-chain fatty acid or a product of the branched-chain fatty acid.Also provided is a cell comprising (i) an exogenous or overexpressedpolynucleotide comprising a nucleic acid sequence encoding an acyltransferase lacking polyketide synthesis activity, and (ii) an exogenousor overexpressed polynucleotide comprising a nucleic acid sequenceencoding a propionyl-CoA carboxylase and/or an exogenous oroverexpressed polynucleotide comprising a nucleic acid sequence encodinga methylmalonyl-CoA mutase, which are expressed in the cell. The cellproduces more branched-chain fatty acid comprising a methyl on one ormore even number carbons than an otherwise similar cell that does notcomprise the polynucleotide(s).

The following numbered paragraphs each succinctly define one or moreexemplary variations of the invention:

1. A method for producing branched-chain fatty acid comprising a methylon one or more even number carbons, the method comprising culturing acell comprising

(aa) an exogenous or overexpressed polynucleotide comprising a nucleicacid sequence encoding a polypeptide that catalyzes the conversion ofpropionyl-CoA to methylmalonyl-CoA and/or (bb) an exogenous oroverexpressed polynucleotide comprising a nucleic acid sequence encodinga polypeptide that catalyzes the conversion of succinyl-CoA tomethylmalonyl-CoA, under conditions allowing expression of thepolynucleotide(s) and production of branched-chain fatty acid, whereinthe cell produces more fatty acid comprising a methyl on one or moreeven number carbons than an otherwise similar cell that does notcomprise the polynucleotide(s).

2. The method of paragraph 1 further comprising extracting from culturethe branched-chain fatty acid or a product of the branched-chain fattyacid.

3. The method of paragraph 1 or paragraph 2, wherein the polypeptidethat catalyzes the conversion of propionyl-CoA to methylmalonyl-CoA is apropionyl-CoA carboxylase and/or the polypeptide that catalyzes theconversion of succinyl-CoA to methylmalonyl-CoA is a methylmalonyl-CoAmutase.

4. The method of paragraph 3, wherein (i) the propionyl-CoA carboxylaseis Streptomyces coelicolor PccB and AccA1 or PccB and AccA2 and/or (ii)the methylmalonyl-CoA mutase is Janibacter sp. HTCC2649methylmalonyl-CoA mutase, S. cinnamonensis MutA and MutB, or E. coliSbm.

5. The method of paragraph 3, wherein (i) the methylmalonyl-CoA mutasecomprises an amino acid sequence having at least about 80% sequenceidentity to the amino acid sequence set forth in SEQ ID NOs: 3, 4, or 28and/or (ii) the propionyl-CoA carboxylase comprises an amino acidsequence having at least about 80% sequence identity to the amino acidsequence set forth in SEQ ID NOs: 9 and 10.

6. The method of any one of paragraphs 3-5, wherein the cell comprisesan exogenous or overexpressed polynucleotide comprising a nucleic acidsequence encoding a methylmalonyl-CoA mutase and further comprises anexogenous or overexpressed polynucleotide comprising a nucleic acidsequence encoding a methylmalonyl-CoA epimerase.

7. The method of any one of paragraphs 1-6, wherein the cell furthercomprises an exogenous or overexpressed polynucleotide encoding an acyltransferase lacking polyketide synthesis activity and/or an exogenous oroverexpressed polynucleotide comprising a nucleic acid sequence encodinga thioesterase.

8. The method of paragraph 7, wherein the acyl transferase is FabD, anacyl transferase domain of a polyketide synthase, or an acyl transferasedomain of Mycobacterium mycocerosic acid synthase.

9. The method of any one of paragraphs 1-8, wherein the cell has beenmodified to attenuate endogenous methylmalonyl-CoA mutase activity,endogenous methylmalonyl-CoA decarboxylase activity, and/or endogenousacyl transferase activity.

10. The method of any one of paragraphs 1-9, wherein the cell produces aType II fatty acid synthase.

11. The method of any one of paragraphs 1-10, wherein the cell isEscherichia coli.

12. A branched-chain fatty acid produced by the method of any one ofparagraphs 1-11.

13. A cell comprising: (i) an exogenous or overexpressed polynucleotidecomprising a nucleic acid sequence encoding an acyl transferase lackingpolyketide synthesis activity, and (ii) an exogenous or overexpressedpolynucleotide comprising a nucleic acid sequence encoding apropionyl-CoA carboxylase and/or an exogenous or overexpressedpolynucleotide comprising a nucleic acid sequence encoding amethylmalonyl-CoA mutase, wherein the polynucleotide(s) are expressedand the cell produces more branched-chain fatty acid comprising a methylon one or more even number carbons than an otherwise similar cell thatdoes not comprise the polynucleotide(s).

14. The cell of paragraph 13, wherein (i) the propionyl-CoA carboxylaseis Streptomyces coelicolor PccB and AccA1 or PccB and AccA2 and/or (ii)the methylmalonyl-CoA mutase is Janibacter sp. HTCC2649methylmalonyl-CoA mutase, S. cinnamonensis MutA and MutB, or E. coliSbm.

15. The cell of paragraph 13, wherein (i) the methylmalonyl-CoA mutasecomprises an amino acid sequence having at least about 80% sequenceidentity to the amino acid sequence set forth in SEQ ID NOs: 3, 4, or 28and/or (ii) the propionyl-CoA carboxylase comprises an amino acidsequence having at least about 80% sequence identity to the amino acidsequence set forth in SEQ ID NOs: 9 and 10.

16. The cell of any one of paragraphs 13-15, wherein the cell comprisesan exogenous or overexpressed polynucleotide comprising a nucleic acidsequence encoding a methylmalonyl-CoA mutase and further comprises anexogenous or overexpressed polynucleotide comprising a nucleic acidsequence encoding a methylmalonyl-CoA epimerase.

17. The cell of any one of paragraphs 13-16, wherein the acyltransferase is FabD, an acyl transferase domain of a polyketidesynthase, or an acyl transferase domain of Mycobacterium mycocerosicacid synthase.

18. The cell of any one of paragraphs 13-17, wherein the cell furthercomprises an exogenous or overexpressed polynucleotide comprises anucleic acid sequence encoding a thioesterase.

19. The cell of any one of paragraphs 13-18, wherein the cell has beenmodified to attenuate endogenous methylmalonyl-CoA mutase activity,endogenous methylmalonyl-CoA decarboxylase activity, and/or endogenousacyl transferase activity.

20. The cell of any one of paragraphs 13-19, wherein the cell isEscherichia coli.

21. A method for producing branched-chain fatty acids in a cellcomprising: a. expressing in the cell one or more recombinantpolypeptides that catalyze the conversion of methylmalonyl-CoA tomethylmalonyl-ACP; and b. culturing the cell under conditions suitablefor producing the polypeptide, such that branched-chain fatty acids areproduced.

22. The method of paragraph 21, wherein the polypeptide is an acyltransferase.

23. The method of paragraph 21, wherein the polypeptide is encoded byfabD.

24. The method of paragraph 22, wherein the polypeptide is a polyketidesynthase or a portion thereof.

25. The method of paragraph 21, wherein the polypeptide is aMycobacterium mycocerosic acid synthase or a portion thereof

26. The method of paragraph 21, wherein the polypeptide has at leastabout 60% sequence identity to a sequence set forth in SEQ ID NO: 19.

27. The method of paragraph 21, wherein the method further includesexpressing in the cell a polypeptide that encodes an exogenousthioesterase.

28. The method of paragraph 21, wherein the cell is an Escherichia cell.

29. The method of paragraph 21, wherein the cell produces higher levelsof branched-chain fatty acids after expression of the polypeptide thanit did prior to expression of the polypeptide.

30. The method of paragraph 21, wherein the branched-chain fatty acidscomprise one or more methyl branches.

31. The method of paragraph 30, wherein the one or more methyl branchesare on even numbered carbons.

32. The method of paragraph 21, wherein the branched-chain fatty acidsare not naturally produced in the cell.

33. Branched-chain fatty acids produced by the method of paragraph 21.

34. A cell comprising at least one recombinant polypeptide thatcatalyzes the conversion of methylmalonyl-CoA to methylmalonyl-ACP,wherein the cell comprising the recombinant polypeptide produces morebranched-chain fatty acids than an otherwise similar cell that does notcomprise the recombinant polypeptide.

35. A method for producing branched-chain fatty acids in a cellcomprising: a. expressing in the cell one or more recombinantpolypeptides that increase the production of methylmalonyl-CoA in thecell; and b. culturing the cell under conditions suitable for producingthe recombinant polypeptide, such that branched-chain fatty acids areproduced.

36. The method of paragraph 35, wherein expression of the polypeptideresults in increased propionyl-CoA synthetase activity in the cell.

37. The method of paragraph 35, wherein the polypeptide haspropionyl-CoA carboxylase activity.

38. The method of paragraph 35, wherein the polypeptide has at leastabout 60% sequence identity to a sequence set forth in SEQ ID NO: 9 orSEQ ID NO: 10.

39. The method of paragraph 35, wherein the method further includesexpressing in the cell a polypeptide that encodes an exogenousthioesterase.

40. The method of paragraph 35, wherein the cell is an Escherichia cell.

41. The method of paragraph 35, wherein the cell produces higher levelsof branched-chain fatty acids after expression of the polypeptide thanit did prior to expression of the polypeptide.

42. The method of paragraph 35, wherein the branched-chain fatty acidscomprise one or more methyl branches.

43. The method of paragraph 42, wherein the one or more methyl branchesare on even numbered carbons.

44. The method of paragraph 35, wherein the branched-chain fatty acidsare not naturally produced in the cell.

45. Branched-chain fatty acids produced by the method of paragraph 35.

46. A cell comprising at least one recombinant polypeptide thatincreases the production of methylmalonyl-CoA in the cell, wherein thecell comprising the recombinant polypeptide produces more branched-chainfatty acids than an otherwise similar cell that does not comprise therecombinant polypeptide.

47. A method for producing branched-chain fatty acids in a cellcomprising: a. expressing in the cell a polypeptide that haspropionyl-CoA synthetase activity; b. inhibiting propionylation of thepropionyl-CoA synthetase; and c. culturing the cell under conditionssuitable for producing the polypeptide, such that branched-chain fattyacids are produced.

48. The method of paragraph 47, wherein the polypeptide does not includea lysine that is subject to propionylation.

49. The method of paragraph 47, wherein step c) includes providing asource of resveratrol into a culture medium used to culture the cell.

50. The method of paragraph 47, wherein the cell does not include anN-acetyltransferase enzyme responsible for propionylation of thepropionyl-CoA synthetase.

51. The method of paragraph 47, wherein the polypeptide has at leastabout 60% sequence identity to the protein encoded by SEQ ID NO: 22.

52. The method of paragraph 47, wherein the cell contains increasedenzymatic activity for removal of propionyl groups from one or morelysine residues of propionyl-CoA synthetase.

53. The method of paragraph 47, wherein the method further includesexpressing in the cell a polypeptide that encodes an exogenousthioesterase.

54. The method of paragraph 47, wherein the cell is an Escherichia cell.

55. The method of paragraph 47, wherein the cell produces higher levelsof branched-chain fatty acids after expression of the polypeptide thanit did prior to expression of the polypeptide.

56. The method of paragraph 47, wherein the branched-chain fatty acidscomprise one or more methyl branches.

57. The method of paragraph 56, wherein the one or more methyl branchesare on even numbered carbons.

58. The method of paragraph 47, wherein the branched-chain fatty acidsare not naturally produced in the cell.

59. Branched-chain fatty acids produced by the method of paragraph 47.

60. A method for producing branched-chain fatty acids in a cellcomprising: a. expressing in the cell a polypeptide that hasmethylmalonyl-CoA mutase activity; b. expressing in a cell a polypeptidethat has methylmalonyl-CoA epimerase activity; and c. culturing the cellunder conditions suitable for producing the polypeptides, such thatbranched-chain fatty acids are produced.

61. The method of paragraph 60, wherein the methylmalonyl-CoA mutasepolypeptide has at least about 60% sequence identity to a sequence setforth in SEQ ID NO: 3 or SEQ ID NO: 4.

62. The method of paragraph 60, wherein the methylmalonyl-CoA epimerasepolypeptide has at least about 60% sequence identity to a sequence setforth in SEQ ID NO: 6.

63. The method of paragraph 60, wherein the method further includesexpressing in the cell a polypeptide that encodes an exogenousthioesterase.

64. The method of paragraph 60, wherein the cell is an Escherichia cell.

65. The method of paragraph 60, wherein the cell produces higher levelsof branched-chain fatty acids after expression of the polypeptide thanit did prior to expression of the polypeptide.

66. The method of paragraph 60, wherein the branched-chain fatty acidscomprise one or more methyl branches.

67. The method of paragraph 66, wherein the one or more methyl branchesare on even numbered carbons.

68. The method of paragraph 60, wherein the branched-chain fatty acidsare not naturally produced in the cell.

69. Branched-chain fatty acids produced by the method of paragraph 60.

70. A cell comprising recombinant polypeptides having methylmalonyl-CoAmutase activity and methylmalonyl-CoA epimerase activity, wherein thecell comprising the recombinant polypeptides produces morebranched-chain fatty acids than an otherwise similar cell that does notcomprise the recombinant polypeptide.

71. A composition comprising a mixture of biologically-producedbranched-chain fatty acids, the branched-chain fatty acids having achain length of C12 to C16 and from about 1 to about 3 methyl branchespositioned on one or more even-numbered carbons.

72. A method for producing branched-chain fatty acids in a cellcomprising: a. expressing in the cell one or more recombinantpolypeptides that increase the production of methylmalonyl-CoA in thecell; b. expressing in the cell a recombinant polypeptide that catalyzesthe conversion of methylmalonyl-CoA to methylmalonyl-ACP; and c.culturing the cell under conditions suitable for producing therecombinant polypeptide, such that branched-chain fatty acids areproduced.

73. The method of paragraph 72, wherein the cell has a deletion in agene for a methylmalonyl-CoA decarboxylase.

74. The method of paragraph 72, wherein the cell additionally produces arecombinant polypeptide with a 3-ketoacyl-ACP synthase activity thatrecognizes methylmalonyl-ACP as a substrate.

75. A method for producing branched-chain fatty acids in a cellcomprising: a. expressing in the cell one or more recombinantpolypeptides that increase the production of methylmalonyl-CoA in thecell; b. expressing in the cell a recombinant polypeptide that catalyzesthe conversion of methylmalonyl-CoA to methylmalonyl-ACP; c. expressingin the cell a recombinant thioesterase; and d. culturing the cell underconditions suitable for producing the recombinant polypeptide, such thatbranched-chain fatty acids are produced.

76. The method of paragraph 75, wherein the cell has a deletion in agene for a methylmalonyl-CoA decarboxylase.

77. The method of paragraph 75, wherein the cell additionally produces arecombinant polypeptide with a 3-ketoacyl-ACP synthase activity thatrecognizes methylmalonyl-ACP as a substrate.

78. A method for producing branched-chain fatty acids in a cell, thebranched-chain fatty acids having a chain length from about 10 to 18carbons and branching at the second carbon, the method comprising: a.modifying the cell to increase carbon flow to methylmalonyl-CoA; and b.culturing the cell under conditions suitable for carbon flow tomethylmalonyl-CoA to be increased, such that branched-chain fatty acidshaving a chain length from about 10 to about 18 carbons and branching atthe second carbon are produced.

79. The method of paragraph 78, wherein the branching at the secondcarbon is a methyl branch.

80. A method for producing branched-chain fatty acids in a cell, thebranched-chain fatty acids having a chain length from about 10 to 18carbons and branching at the fourth carbon, the method comprising: a.modifying the cell to increase carbon flow to methylmalonyl-CoA; and b.culturing the cell under conditions suitable for carbon flow tomethylmalonyl-CoA to be increased, such that branched-chain fatty acidshaving a chain length from about 10 to about 18 carbons and branching atthe fourth carbon are produced.

81. The method of paragraph 80, wherein the branching at the fourthcarbon is a methyl branch.

82. A method for producing branched-chain fatty acids in a cell, thebranched-chain fatty acids having a chain length from about 10 to 18carbons and branching at the sixth carbon, the method comprising: a.modifying the cell to increase carbon flow to methylmalonyl-CoA; and b.culturing the cell under conditions suitable for carbon flow tomethylmalonyl-CoA to be increased, such that branched-chain fatty acidshaving a chain length from about 10 to about 18 carbons and branching atthe sixth carbon are produced.

83. The method of paragraph 82, wherein the branching at the sixthcarbon is a methyl branch.

84. A method for producing branched-chain fatty acids in a cell, thebranched-chain fatty acids having a chain length from about 12 to 18carbons and branching at the eighth carbon, the method comprising: a.modifying the cell to increase carbon flow to methylmalonyl-CoA; and b.culturing the cell under conditions suitable for carbon flow tomethylmalonyl-CoA to be increased, such that branched-chain fatty acidshaving a chain length from about 12 to about 18 carbons and branching atthe eighth carbon are produced.

85. The method of paragraph 84, wherein the branching at the eighthcarbon is a methyl branch.

86. A method for producing branched-chain fatty acids in a cell, thebranched-chain fatty acids having a chain length from about 14 to 18carbons and branching at the tenth carbon, the method comprising: a.modifying the cell to increase carbon flow to methylmalonyl-CoA; and b.culturing the cell under conditions suitable for carbon flow tomethylmalonyl-CoA to be increased, such that branched-chain fatty acidshaving a chain length from about 14 to about 18 carbons and branching atthe tenth carbon are produced.

87. The method of paragraph 86, wherein the branching at the tenthcarbon is a methyl branch.

88. A method for producing branched-chain fatty acids in a cell, thebranched-chain fatty acids having a chain length from about 16 to 18carbons and branching at the twelfth carbon, the method comprising: a.modifying the cell to increase carbon flow to methylmalonyl-CoA; and b.culturing the cell under conditions suitable for carbon flow tomethylmalonyl-CoA to be increased, such that branched-chain fatty acidshaving a chain length from about 16 to about 18 carbons and branching atthe twelfth carbon are produced.

89. The method of paragraph 88, wherein the branching at the twelfthcarbon is a methyl branch.

90. A method for producing branched-chain fatty acids in a cell, thebranched-chain fatty acids having a chain length from about 10 to 18carbons and branching at the second carbon, the method comprising: a.modifying the cell to generate methylmalonyl-ACP from methylmalonyl-CoA;and b. culturing the cell under conditions suitable for generation ofmethylmalonyl-ACP from methylmalonyl-CoA, such that branched-chain fattyacids having a chain length from about 10 to about 18 carbons andbranching at the second carbon are produced.

91. The method of paragraph 90, wherein the branching at the secondcarbon is a methyl branch.

92. A method for producing branched-chain fatty acids in a cell, thebranched-chain fatty acids having a chain length from about 10 to 18carbons and branching at the fourth carbon, the method comprising: a.modifying the cell to generate methylmalonyl-ACP from methylmalonyl-CoA;and b. culturing the cell under conditions suitable for generation ofmethylmalonyl-ACP from methylmalonyl-CoA, such that branched-chain fattyacids having a chain length from about 10 to about 18 carbons andbranching at the fourth carbon are produced.

93. The method of paragraph 92, wherein the branching at the fourthcarbon is a methyl branch.

94. A method for producing branched-chain fatty acids in a cell, thebranched-chain fatty acids having a chain length from about 10 to 18carbons and branching at the sixth carbon, the method comprising: a.modifying the cell to generate methylmalonyl-ACP from methylmalonyl-CoA;and b. culturing the cell under conditions suitable for generation ofmethylmalonyl-ACP from methylmalonyl-CoA, such that branched-chain fattyacids having a chain length from about 10 to about 18 carbons andbranching at the sixth carbon are produced.

95. The method of paragraph 94, wherein the branching at the sixthcarbon is a methyl branch.

96. A method for producing branched-chain fatty acids in a cell, thebranched-chain fatty acids having a chain length from about 12 to 18carbons and branching at the eighth carbon, the method comprising: a.modifying the cell to generate methylmalonyl-ACP from methylmalonyl-CoA;and b. culturing the cell under conditions suitable for generation ofmethylmalonyl-ACP from methylmalonyl-CoA, such that branched-chain fattyacids having a chain length from about 12 to about 18 carbons andbranching at the eighth carbon are produced.

97. The method of paragraph 96, wherein the branching at the eighthcarbon is a methyl branch.

98. A method for producing branched-chain fatty acids in a cell, thebranched-chain fatty acids having a chain length from about 14 to 18carbons and branching at the tenth carbon, the method comprising: a.modifying the cell to generate methylmalonyl-ACP from methylmalonyl-CoA;and b. culturing the cell under conditions suitable for generation ofmethylmalonyl-ACP from methylmalonyl-CoA, such that branched-chain fattyacids having a chain length from about 14 to about 18 carbons andbranching at the tenth carbon are produced.

99. The method of paragraph 98, wherein the branching at the tenthcarbon is a methyl branch.

100. A method for producing branched-chain fatty acids in a cell, thebranched-chain fatty acids having a chain length from about 16 to 18carbons and branching at the twelfth carbon, the method comprising: a.modifying the cell to generate methylmalonyl-ACP from methylmalonyl-CoA;and b. culturing the cell under conditions suitable for generation ofmethylmalonyl-ACP from methylmalonyl-CoA, such that branched-chain fattyacids having a chain length from about 16 to about 18 carbons andbranching at the twelfth carbon are produced.

101. The method of paragraph 100, wherein the branching at the twelfthcarbon is a methyl branch.

102. A method for producing modified fatty acids in a cell comprising:a. providing a cell having type II fatty acid synthase activity; b.expressing in the cell one or more recombinant polypeptides thatcatalyze formation of at least one intermediate metabolite, wherein theat least one intermediate metabolite is incorporated by the type IIfatty acid synthase; and c. culturing the cell under conditions suitablefor producing the recombinant polypeptide, such that modified fattyacids are produced.

103. The method of paragraph 102, wherein the cell is an Escherichiacell.

104. The method of paragraph 102, wherein the intermediate metabolite ismethylmalonyl-ACP.

105. The method of paragraph 102, wherein the polypeptide(s) catalyzethe conversion of methylmalonyl-CoA to methylmalonyl-ACP.

106. The method of paragraph 102, wherein the cell produces higherlevels of modified fatty acids after expression of the polypeptide thanit did prior to expression of the polypeptide.

107. The method of paragraph 102, wherein the modified fatty acidscomprise one or more methyl branches on even-numbered carbons.

108. The method of paragraph 102, wherein the polypeptide is an acyltransferase.

109. The method of paragraph 102, wherein the polypeptide is encoded byfabD.

110. The method of paragraph 102, wherein the polypeptide is apolyketide synthase or a portion thereof.

111. The method of paragraph 102, wherein the polypeptide is aMycobacterium mycocerosic acid synthase or a portion thereof.

112. An Escherichia cell that produces branched-chain fatty acids havinga chain length from about 10 to about 18 carbons and comprising one ormore methyl branches on one or more even-numbered carbons.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a mutA nucleotide sequence (SEQ ID NO: 1).

FIG. 2 is a mutB nucleotide sequence (SEQ ID NO: 2).

FIG. 3 is a MutA protein sequence (SEQ ID NO: 3).

FIG. 4 is a MutB protein sequence (SEQ ID NO: 4).

FIG. 5 is a methylmalonyl-CoA epimerase nucleotide sequence (SEQ ID NO:5).

FIG. 6 is a methylmalonyl-CoA epimerase protein sequence (SEQ ID NO: 6).

FIG. 7 is a DNA sequence for accA1 (GenBank Accession No. AF113603.1)(SEQ ID NO: 7).

FIG. 8 is a DNA sequence for pccB (GenBank Accession No. AF113605.1)(SEQ ID NO: 8).

FIG. 9 is a protein sequence for AccA1 (SEQ ID NO: 9).

FIG. 10 is a protein sequence for PccB (SEQ ID NO: 10).

FIG. 11 shows element 1 including the P_(Llac0-1) sequence and the phageT7 gene10 ribosome binding site (SEQ ID NO: 11).

FIG. 12 shows element 2 including the optimized accA1 gene sequence (SEQID NO: 12).

FIG. 13 shows element 3 including the spacer sequence (SEQ ID NO: 13).

FIG. 14 shows element 4 including the optimized pccB sequence (SEQ IDNO: 14).

FIG. 15 is a synthetic sequence for propionyl-CoA carboxylase geneexpression (SEQ ID NO: 15).

FIG. 16 is the forward primer sequence for PrpE (SEQ ID NO: 16).

FIG. 17 is the reverse primer sequence for PrpE (SEQ ID NO: 17).

FIG. 18 is the MMAT domain sequence from Mycobacterium bovis BCG (SEQ IDNO: 18).

FIG. 19 is a protein sequence for the Mycobacterium bovis BCG MAS(GenBank Accession No. YP_(—)979046) (SEQ ID NO: 19).

FIG. 20 is a codon-optimized MMAT domain DNA sequence from Mycobacteriumbovis BCG (SEQ ID NO: 20).

FIG. 21 is an alignment of a codon-optimized MMAT domain fromMycobacterium bovis BCG with the original sequence (SEQ ID NOs: 20 and21).

FIG. 22 is the protein sequence of Salmonella enterica propionyl CoAsynthase PrpE (GenBank Accession No. AAC44817) (SEQ ID NO: 22).

FIG. 23 is the DNA sequence of Salmonella enterica propionyl CoAsynthase PrpE (SEQ ID NO. 23).

FIG. 24 is a bar graph illustrating methylmalonyl-CoA production (ng/ml)in E. coli strain K27-Z1 harboring pTrcHisA pZA31 (control), pZA31 mutABSs epi (MutAB Epi), pTrcHisA Ec sbm (Sbm), or pTrcHisA Ec sbm pZA31 Mbmmat (Sbm/Mmat). No methylmalonyl-CoA was identified in the controlsample; the figure indicates the background level of detection.

FIG. 25 is a bar graph illustrating methylmalonyl-CoA production (ng/ml)in E. coli BW25113 (control) and BW25113 harboring pZA31-accA1-pccB(Pcc). No methylmalonyl-CoA was identified in the control sample; thefigure indicates the background level of detection. Two biologicalreplicates are represented.

FIG. 26 is a two-dimensional (2D) representation of the 2D Total IonChromatogram resulting from a sample of fatty acid produced by BL21 Star(DE3) E. coli harboring pTrcHisA Ec sbm So ce epi pZA31 mmat. Lightareas on the figure indicate the presence of sample material. Peak namesand arrows indicate samples that were further characterized by massspectrometry.

FIG. 27 is a two-dimensional (2D) representation of the 2D Total IonChromatogram resulting from a sample produced by a control strain, BL21Star (DE3) E. coli harboring pTrcHisA pZA31. No branched-chain fattyacid was detected. Arrows indicate the presence of straight-chain fattyacid derivatives of the indicated chain length.

FIG. 28 is a representation of the mass spectra of peaks 54, 55, and 57identified in FIG. 26. Eight- and ten-carbon branched-chain fatty acidsare depicted in the top two profiles and were identified by the almostcomplete absence of the circled fragment. A twelve-branched fatty acidwas tentatively identified and is depicted in the third profile.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to improved biological production of scatteredbranched-chain fatty acids. In addition, in certain embodiments, theinvention provides improved compositions of biologically producedscattered branched-chain fatty acids having defined chain lengths withmethyl branches at one or more even-numbered carbons within the fattyacid. In addition, in certain embodiments, the fatty acid length can betailored to a predetermined length, such as, for example, to producefatty acids with a backbone of C12 to C16. In certain embodiments, themethods and/or cells can produce a mixture of fatty acids having variednumbers of methyl branches, varied positions of the methyl branches, andvaried length of the fatty acids, such as, for example, a mixture offatty acids having a chain length of C12 to C16 and from about 0 toabout 3 methyl branches positioned on one or more even-numbered carbons.

As used herein, “amplify,” “amplified,” or “amplification” refers to anyprocess or protocol for copying a polynucleotide sequence into a largernumber of polynucleotide molecules, e.g., by reverse transcription,polymerase chain reaction, and ligase chain reaction.

As used herein, an “antisense sequence” refers to a sequence thatspecifically hybridizes with a second polynucleotide sequence. Forinstance, an antisense sequence is a DNA sequence that is invertedrelative to its normal orientation for transcription. Antisensesequences can express an RNA transcript that is complementary to atarget mRNA molecule expressed within the host cell (e.g., it canhybridize to target mRNA molecule through Watson-Crick base pairing).

As used herein, “cDNA” refers to a DNA that is complementary oridentical to an mRNA, in either single stranded or double stranded form.

As used herein, the carbons in fatty acids are numbered with the firstcarbon as part of the carboxylic acid group, and the second carbon (C2)adjacent to the first. The numbers continue so that the highest numbercarbon is farthest from the carboxylic acid group. “Even number” carbonsinclude C2, C4, C6, C8, C10, C12, C14, and so on.

As used herein, “complementary” refers to a polynucleotide that can basepair with a second polynucleotide. Put another way, “complementary”describes the relationship between two single-stranded nucleic acidsequences that anneal by base-pairing. For example, a polynucleotidehaving the sequence 5′-GTCCGA-3′ is complementary to a polynucleotidewith the sequence 5′-TCGGAC-3′.

As used herein, a “conservative substitution” refers to the substitutionin a polypeptide of an amino acid with a functionally similar aminoacid. Put another way, a conservative substitution involves replacementof an amino acid residue with an amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined within the art, and include amino acids with basicside chains (e.g., lysine, arginine, and histidine), acidic side chains(e.g., aspartic acid and glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, andcysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, and tryptophan),beta-branched side chains (e.g., threonine, valine, and isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, andhistidine).

As used herein, “encoding” refers to the inherent property ofnucleotides to serve as templates for synthesis of other polymers andmacromolecules. Unless otherwise specified, a “nucleotide sequenceencoding an amino acid sequence” includes all nucleotide sequences thatare degenerate versions of each other and that encode the same aminoacid sequence.

As used herein, “endogenous” refers to polynucleotides, polypeptides, orother compounds that are expressed naturally or originate within anorganism or cell. That is, endogenous polynucleotides, polypeptides, orother compounds are not exogenous. For instance, an “endogenous”polynucleotide or peptide is present in the cell when the cell wasoriginally isolated from nature.

As used herein, “expression vector” refers to a vector comprising arecombinant polynucleotide comprising expression control sequencesoperatively linked to a nucleotide sequence to be expressed. Forexample, suitable expression vectors include, without limitation,autonomously replicating vectors or vectors integrated into thechromosome. In some instances, an expression vector is a viral-basedvector.

As used herein, “exogenous” refers to any polynucleotide or polypeptidethat is not naturally expressed or produced in the particular cell ororganism where expression is desired. Exogenous polynucleotides,polypeptides, or other compounds are not endogenous.

As used herein, “hybridization” includes any process by which a strandof a nucleic acid joins with a complementary nucleic acid strand throughbase-pairing. Thus, the term refers to the ability of the complement ofthe target sequence to bind to a test (i.e., target) sequence, orvice-versa.

As used herein, “hybridization conditions” are typically classified bydegree of “stringency” of the conditions under which hybridization ismeasured. The degree of stringency can be based, for example, on themelting temperature (T_(m)) of the nucleic acid binding complex orprobe. For example, “maximum stringency” typically occurs at aboutT_(m)-5° C. (5° below the T_(m) of the probe); “high stringency” atabout 5-10° C. below the T_(m); “intermediate stringency” at about10-20° below the T_(m) of the probe; and “low stringency” at about20-25° C. below the T. Alternatively, or in addition, hybridizationconditions can be based upon the salt or ionic strength conditions ofhybridization and/or one or more stringency washes. For example,6×SSC=very low stringency; 3×SSC=low to medium stringency; 1×SSC=mediumstringency; and 0.5×SSC=high stringency. Functionally, maximumstringency conditions may be used to identify nucleic acid sequenceshaving strict (i.e., about 100%) identity or near-strict identity withthe hybridization probe; while high stringency conditions are used toidentify nucleic acid sequences having about 80% or more sequenceidentity with the probe.

As used herein, “identical” or percent “identity” in the context of twoor more polynucleotide or polypeptide sequences refers to two or moresequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned for maximum correspondence, as measured using sequencecomparison algorithms or by visual inspection.

As used herein, “long-chain fatty acids” refers to fatty acids withaliphatic tails longer than 14 carbons. In some embodiments of theinvention, long-chain fatty acids are provided that comprise 15, 16, 17,18, 19, 20, 21, or 22 carbons in the carbon backbone.

As used herein, “medium-chain fatty acids” refers to fatty acids withaliphatic tails between 6 and 14 carbons. In certain embodiments, themedium-chain fatty acids can have from 11 to 13 carbons.

As used herein, “naturally-occurring” refers to an object that can befound in nature. For example, a polypeptide or polynucleotide sequencethat is present in an organism (including viruses) that can be isolatedfrom a source in nature and which has not been intentionally modified byman in the laboratory is naturally-occurring.

As used herein, “operably linked,” when describing the relationshipbetween two DNA regions or two polypeptide regions, means that theregions are functionally related to each other. For example, a promoteris operably linked to a coding sequence if it controls the transcriptionof the sequence; a ribosome binding site is operably linked to a codingsequence if it is positioned so as to permit translation; and a signalsequence is operably linked to a peptide if it functions as a signalsequence, such as by participating in the secretion of the mature formof the protein.

As used herein, “overexpression” refers to expression of apolynucleotide to produce a product (e.g., a polypeptide or RNA) at ahigher level than the polynucleotide is normally expressed in the hostcell. An overexpressed polynucleotide is generally a polynucleotidenative to the host cell, the product of which is generated in a greateramount than that normally found in the host cell. Overexpression isachieved by, for instance and without limitation, operably linking thepolynucleotide to a different promoter than the polynucleotide's nativepromoter or introducing additional copies of the polynucleotide into thehost cell.

As used herein, “polynucleotide” refers to a polymer composed ofnucleotides. The polynucleotide may be in the form of a separatefragment or as a component of a larger nucleotide sequence construct,which has been derived from a nucleotide sequence isolated at least oncein a quantity or concentration enabling identification, manipulation,and recovery of the sequence and its component nucleotide sequences bystandard molecular biology methods, for example, using a cloning vector.When a nucleotide sequence is represented by a DNA sequence (i.e., A, T,G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which“U” replaces “T.” Put another way, “polynucleotide” refers to a polymerof nucleotides removed from other nucleotides (a separate fragment orentity) or can be a component or element of a larger nucleotideconstruct, such as an expression vector or a polycistronic sequence.Polynucleotides include DNA, RNA and cDNA sequences.

As used herein, “polypeptide” refers to a polymer composed of amino acidresidues which may or may not contain modifications such as phosphatesand formyl groups.

As used herein, “recombinant expression vector” refers to a DNAconstruct used to express a polynucleotide that encodes a desiredpolypeptide. A recombinant expression vector can include, for example, atranscriptional subunit comprising (i) an assembly of genetic elementshaving a regulatory role in gene expression, for example, promoters andenhancers, (ii) a structural or coding sequence which is transcribedinto mRNA and translated into protein, and (iii) appropriatetranscription and translation initiation and termination sequences.Recombinant expression vectors are constructed in any suitable manner.The nature of the vector is not critical, and any vector may be used,including plasmid, virus, bacteriophage, and transposon. Possiblevectors for use in the invention include, but are not limited to,chromosomal, nonchromosomal and synthetic DNA sequences, e.g., bacterialplasmids; phage DNA; yeast plasmids; and vectors derived fromcombinations of plasmids and phage DNA, DNA from viruses such asvaccinia, adenovirus, fowl pox, baculovirus, SV40, and pseudorabies.

As used herein, “primer” refers to a polynucleotide that is capable ofspecifically hybridizing to a designated polynucleotide template andproviding a point of initiation for synthesis of a complementarypolynucleotide when the polynucleotide primer is placed under conditionsin which synthesis is induced.

As used herein, “recombinant polynucleotide” refers to a polynucleotidehaving sequences that are not naturally joined together. A recombinantpolynucleotide may be included in a suitable vector, and the vector canbe used to transform a suitable host cell. A host cell that comprisesthe recombinant polynucleotide is referred to as a “recombinant hostcell.” The polynucleotide is then expressed in the recombinant host cellto produce, e.g., a “recombinant polypeptide.”

As used herein, “specific hybridization” refers to the binding,duplexing, or hybridizing of a polynucleotide preferentially to aparticular nucleotide sequence under stringent conditions.

As used herein, “stringent conditions” refers to conditions under whicha probe will hybridize preferentially to its target subsequence, and toa lesser extent to, or not at all to, other sequences.

As used herein, “short-chain fatty acids” refers to fatty acids havingaliphatic tails with fewer than 6 carbons.

As used herein, “substantially homologous” or “substantially identical”in the context of two nucleic acids or polypeptides, generally refers totwo or more sequences or subsequences that have at least 40%, 60%, 80%,90%, 95%, 96%, 97%, 98% or 99% nucleotide or amino acid residueidentity, when compared and aligned for maximum correspondence, asmeasured using sequence comparison algorithms or by visual inspection.The substantial identity can exist over any suitable region of thesequences, such as, for example, a region that is at least about 50residues in length, a region that is at least about 100 residues, or aregion that is at least about 150 residues. In certain embodiments, thesequences are substantially identical over the entire length of eitheror both comparison biopolymers.

In one embodiment, the invention relates to a novel method of producingscattered branched-chain fatty acids (or products derived from scatteredbranched-chain fatty acid) using bacteria. In general, the methodincludes increasing the supply of methylmalonyl-CoA and/or theconversion of methylmalonyl-CoA to methylmalonyl-ACP within the cell,incorporating the branch from the methylmalonyl-CoA into the fatty acid,and, optionally, using a thioesterase to specify the range of size ofthe fatty acids. In certain embodiments, the method providesbranched-chain fatty acids having a chain length of C12 to C16. Inaddition, in certain embodiments, the branched-chain fatty acids havefrom about 0 to about 3 methyl branches, such as from about 1 to about 3methyl branches, such as, for example, from about 1 to about 2 methylbranches, or 1, 2, or 3 methyl branches positioned on one or morecarbons. In certain embodiments, the methyl branches are positioned oneven-numbered carbons.

In one embodiment, scattered branched-chain fatty acid production isincreased by increasing the production of methylmalonyl-CoA within thecell via, e.g., propionyl-CoA and/or succinyl-CoA intermediates. Thus,in one aspect, the invention provides a method for producingbranched-chain fatty acid comprising a methyl on one or more even numbercarbons. The method comprises culturing a cell comprising an exogenousor overexpressed polynucleotide comprising a nucleic acid sequenceencoding a polypeptide that catalyzes the conversion of propionyl-CoA tomethylmalonyl-CoA and/or an exogenous or overexpressed polynucleotidecomprising a nucleic acid sequence encoding a polypeptide that catalyzesthe conversion of succinyl-CoA to methylmalonyl-CoA. The cell iscultured under conditions allowing expression of the polynucleotide(s)and production of the branched-chain fatty acid. The cell produces morebranched-chain fatty acid comprising a methyl branch on one or more evennumber carbons than an otherwise similar cell that does not comprise thepolynucleotide(s) (e.g., a cell of the same cell type or derived fromthe same organism that does not comprise the polynucleotide(s)).Propionyl-CoA is converted to methylmalonyl-CoA by, e.g., the action ofa propionyl-CoA carboxylase. Any propionyl-CoA carboxylase thatcatalyzes the conversion of propionyl-CoA to methylmalonyl-CoA issuitable for use in the inventive method. An exemplary propionyl-CoAcarboxylase is a carboxylase from Streptomyces coelicolor, whichcomprises two heterologous subunits encoded by pccB and by either accA1or accA2. In certain embodiments, the cell of the inventive method isengineered to produce PccB and AccA1 or PccB and AccA2. In one aspect,the cell comprises one or more polynucleotides encoding polypeptide(s)comprising an amino acid sequence at least about 80% identical (e.g.,85%, 90%, 95%, or 100% identical) to the amino acid sequences set forthin SEQ ID NO: 9 and/or 10. Additional, non-limiting examples ofpolypeptides that catalyze the conversion of propionyl-CoA tomethylmalonyl-CoA are propionyl-CoA carboxylases from Mycobacteriumsmegmatis, Homo sapiens, Acinetobacter baumannii, Brucella suis,Saccharopolyspora erythraea, Burkholderia glumae, and Aedes aegypti, aswell as the propionyl-CoA carboxylases set forth in Table A.

TABLE A GenBank Organism Accession Description SEQ ID NO: Ehrlichiachaffeensis YP_507303 Propionyl-CoA carboxylase alpha subunit 51 (PCCA)Ehrlichia chaffeensis YP_507410 Propionyl-CoA carboxylase beta subunit52 (PCCB) Agrobacterium vitis YP_002547482 Propionyl-CoA carboxylasealpha subunit 53 (PCCA) Agrobacterium vitis YP_002547479 Propionyl-CoAcarboxylase beta subunit 54 (PCCB) Methylobacterium YP_003069256Propionyl-CoA carboxylase alpha subunit 55 extorquens (PCCA)Methylobacterium YP_003065890 Propionyl-CoA carboxylase beta subunit 56extorquens (PCCB) Sinorhizobium meliloti NP_437988 Propionyl-CoAcarboxylase alpha subunit 57 (PCCA) Sinorhizobium meliloti NP_437987Propionyl-CoA carboxylase beta subunit 58 (PCCB) Ruegeria pomeroyiYP_166352 Propionyl-CoA carboxylase alpha subunit 59 (PCCA) Ruegeriapomeroyi YP_166345 Propionyl-CoA carboxylase beta subunit 60 (PCCB)

Optionally, the cell is modified to increase carbon flow topropionyl-CoA (and then onward to methylmalonyl-CoA) by, for example,increasing expression of (i.e., overexpressing) prpE or otherpropionyl-CoA synthetase genes. Alternatively or in addition, anexogenous polynucleotide comprising a nucleic acid sequence encoding apropionyl-CoA synthetase is introduced into the host cell to upregulatepropionyl-CoA production. Additionally, feeding host cells (e.g.,microbes) large amounts of methionine, isoleucine, valine, threonine,propionic acid, and/or odd-chain length fatty acids (such as valericacid) increases production of the propionyl-CoA precursor ofmethylmalonyl-CoA.

Methylmalonyl-CoA production via propionyl-CoA also is increasedutilizing the metabolic pathway that converts pyruvate to propionyl-CoA,with lactate, lactoyl-CoA, and acrylyl-CoA as intermediates. Carbon flowto propionyl-CoA is upregulated by overproducing the enzymes of thepathway, producing exogenous enzymes catalyzing one or more conversionsof the pathway, and/or by providing pyruvate or lactate in largeramounts than normally found in the host cell. For example, in anyembodiment of the invention, the cell comprises an exogenous oroverexpressed polynucleotide encoding lactate dehydrogenase, lactate CoAtransferase, lactyl-CoA dehydratase, and/or acrylyl-CoA reductase.

In addition, in any aspect of the invention, carbon flow to branchpathways not contributing to formation of the desired branched-chainfatty acid is minimized by attenuation of endogenous enzyme activityresponsible for the diversion of carbon. Complete abolishment ofendogenous activity is not required; any reduction in activity issuitable in the context of the invention. Enzyme activity is attenuated(i.e., reduced or abolished) by, for example, mutating the codingsequence for the enzyme to create a non-functional or reduced-functionpolypeptide, by removing all or part of the coding sequence for theenzyme from the cellular genome, by interfering with translation of anRNA transcript encoding the enzyme (e.g., using antisenseoligonucleotides), or by manipulating the expression control sequencesinfluencing expression of the enzyme. For example, in one aspect, thecell is modified to prevent methylmalonyl-CoA degradation, therebyincreasing the amount of methylmalonyl-CoA available for conversion tomethylmalonyl-ACP. Methylmalonyl-CoA degradation is reduced by, forexample, deleting or inactivating methylmalonyl-CoA decarboxylase fromthe host. Put another way, the cell is modified to attenuate endogenousmethylmalonyl-CoA decarboxylase activity. In E. coli, for example,methylmalonyl-CoA decarboxylase activity is attenuated by, for example,deleting or mutating ygfG. Optionally, endogenous acyl transferaseactivity is attenuated. Alternatively or in addition, methylmalonyl-CoAproduction within the cell is increased by preventing alternativemetabolism of propionyl-CoA to succinyl-CoA, such as, for example, bydeleting or otherwise reducing (attenuating) the activity of anendogenous methylmalonyl-CoA mutase gene. Optionally, methylmalonyl-CoAlevels are increased by increasing the degradation of valine directly tomethylmalonyl-CoA. Valine degradation comprises the followingintermediates: α-ketoisovalerate, isobutyryl-CoA, methacrylyl-CoA,β-hydroxyisobutyryl-CoA, β-hydroxyisobutyrate, and methylmalonatesemialdehyde. Optionally, methylmalonate semialdehyde is converteddirectly to methylmalonyl-CoA or indirectly through a propionyl-CoAintermediate. In an exemplary embodiment, the cell of the inventioncomprises an overexpressed or exogenous polynucleotide comprising anucleic acid sequence encoding one or more of the following enzymes:L-valine:2-oxoglutarate aminotransferase, 2-oxoisovaleratedehydrogenase, isobutyryl-CoA:FAD oxidoreductase,3-hydroxy-isobutyryl-CoA hydro-lyase, 3-hydroxyisobutyryl-CoA hydrolase,3-hydroxyisobutyrate dehydrogenase, and/or methylmalonate-semialdehydedehydrogenase. Methylmalonate-semialdehyde dehydrogenase catalyzes theproduction of propanoyl-CoA, which can be converted to methylmalonyl-CoAby propanoyl-CoA carboxylase.

In one aspect, the cell comprises an exogenous or overexpressedpolynucleotide comprising a nucleic acid sequence encoding a polypeptidethat catalyzes the conversion of succinyl-CoA to methylmalonyl-CoA. Anexemplary polypeptide that catalyzes the reaction is methylmalonyl-CoAmutase. In any embodiment of the invention, the cell is engineered tooverexpress a methylmalonyl-CoA mutase gene, such as, for example, sbm(encoding Sleeping Beauty mutase) in E. coli. Alternatively or inaddition, an exogenous polynucleotide comprising a nucleic acid sequenceencoding a methylmalonyl-CoA mutase is expressed in the cell. Exemplarymethylmalonyl-CoA mutases include, but are not limited to, Sbm from E.coli, MutA and/or MutB from Streptomyces cinnamonensis, andmethylmalonyl-CoA mutases from Janibacter sp. HTCC2649, Corynebacteriumglutamicum, Euglena gracilis, Homo sapiens, Propionibacterium shermanii,Bacillus megaterium, and Mycobacterium smegmatis. Additional,non-limiting examples of polypeptides that catalyze the conversion ofsuccinyl-CoA to methylmalonyl-CoA are provided in Table B.

TABLE B GenBank Organism Accession Description SEQ ID NO. Bacillusmegaterium YP_003564880 methylmalonyl-CoA mutase small subunit 61 (mutA)Bacillus megaterium YP_003564879 methylmalonyl-CoA mutase large subunit62 (mutB) Mycobacterium YP_001282809 methylmalonyl-CoA mutase smallsubunit 63 tuberculosis (mutA) Mycobacterium YP_001282810methylmalonyl-CoA mutase large subunit 64 tuberculosis (mutB)Corynebacterium YP_225814 methylmalonyl-COA mutase small subunit 65glutamicum (mutA) Corynebacterium YP_225813 methylmalonyl-CoA mutaselarge subunit 66 glutamicum (mutB) Rhodococcus YP_002766535methylmalonyl-CoA mutase small subunit 67 erythropolis (mutA)Rhodococcus YP_002766536 methylmalonyl-CoA mutase large subunit 68erythropolis (mutB) Porphyromonas NP_905776 methylmalonyl-CoA mutasesmall subunit 69 gingivalis (mutA) Porphyromonas NP_905777methylmalonyl-CoA mutase large subunit 70 gingivalis (mutB)

In one aspect, the cell comprises one or more polynucleotides encodingpolypeptide(s) comprising an amino acid sequence at least about 80%identical (e.g., 85%, 90%, 95%, or 100% identical) to the amino acidsequences set forth in SEQ ID NO: 3, 4, and/or 28. The cell can comprisepolynucleotides encoding a methylmalonyl-CoA mutase, a propionyl-CoAcarboxylase, or both.

Depending on the substrate specificity of the fatty acid synthaseproduced by the cell, a methylmalonyl-CoA epimerase also may be desiredto facilitate use of methylmalonyl-CoA as a precursor in fatty acidsynthesis. Thus, in one aspect, the cell further comprises an exogenousor overexpressed polynucleotide comprising a nucleic acid sequenceencoding a methylmalonyl-CoA epimerase. Methylmalonyl-CoA epimerasessuitable for use in the invention include, but are not limited to,Sorangium cellulosum So ce 56 methylmalonyl-CoA epimerase, Streptomycessviceus ATCC 29083 methylmalonyl-CoA epimerase, Kribbella flavida DSM17836 methylmalonyl-CoA epimerase, and methylmalonyl-CoA epimerases fromHomo sapiens, Bacillus megaterium, and Mycobacterium smegmatis.

Production of branched-chain fatty acid comprising a methyl branch onone or more even number carbons also is enhanced by upregulatingconversion of methylmalonyl-CoA to methylmalonyl-ACP. In one or moreembodiments, conversion of methylmalonyl-CoA to methylmalonyl-ACP isincreased in the cell by engineering the cell to produce an acyltransferase (such as the acyl transferase encoded by fabD in E. coli) tocatalyze the formation of methylmalonyl-ACP from methylmalonyl-CoA. Putanother way, in one aspect, the cell further comprises an exogenous oroverexpressed polynucleotide comprising a nucleic acid sequence encodingan acyl transferase. Any suitable acyl transferase can be used, such as,for example and without limitation, an acyl transferase domain from apolyketide synthase, such as those involved in the synthesis ofmonensin, epothilone, amphotericin, candicidin, nystatin, pimaricin,ascomycin, rapamycin, avermiectin, spinosad, mycinamicin, niddamycin,oleandomycin, megalomicin, nanchangmycin, picromycin, rifamycin,oligomycin erythromycin, polyenes, and macrolides, and an acyltransferase domain from Mycobacterium mycocerosic acid synthase. Acyltransferase domains from larger fatty acid synthase enzymes, such asMycobacterium mycocerosic acid synthase, act upon methylmalonyl-CoA inthe absence of other enzymatic domains of the larger synthase.Optionally, the acyl transferase lacks polyketide synthesis activity. By“polyketide synthesis activity” is meant enzymatic activity, other thanacyl transferase activity, that catalyzes the production of polyketidesin a host cell, such as, for example and without limitation,acyltransferase activity, ketoacyl synthase activity, ketoacyl reductaseactivity, dehydratase activity, enoyl reductase activity, acyl carrierprotein activity, and thioesterase activity.

Alternatively, or in addition, in certain embodiments, a 3-ketoacyl-ACPsynthase domain, such as, for example, a domain from a polyketidesynthase or a mycocerosic acid synthase, is added to the fatty acidsynthase of the host cell. In certain embodiments, the host cell (e.g.,microbe) is engineered to include both acyl transferase and3-ketoacyl-ACP synthase domains that can recognize methylmalonyl-CoA. Inaddition, in certain embodiments, genes for the endogenous acyltransferase and/or 3-ketoacyl-ACP synthase activities can be attenuated(e.g., deleted) to minimize the amount of malonyl-CoA incorporation infatty acid synthesis.

In certain embodiments, the invention includes use of a thioesterase tospecify the chain length of the fatty acid, such as, for example, toproduce medium-chain fatty acids. In certain embodiments, the host cellfurther comprises an exogenous or overexpressed polynucleotidecomprising a nucleic acid sequence encoding a thioesterase. In oneaspect, the host cell (e.g., bacteria) is engineered to produce athioesterase that assists in the production of medium-chainbranched-chain fatty acids. Alternatively, the host cell is engineeredto produce (or overproduce) a thioesterase that assists in theproduction of long-chain branched-chain fatty acids. Exemplarythioesterases include, for example, the mallard uropygial glandthioesterase, the California bay thioesterase, the rat mammary glandthioesterase II, E. coli TesA, the Cuphea wrightii thioesterase, andother thioesterases suitable for production of the desired chain-lengthfatty acids.

Optionally, the cell is modified to produce (or increase the productionof) branched acyl-CoA, which is a substrate for elongase in theproduction of long chain fatty acid. In this regard, in an exemplaryembodiment of the invention, the cell comprises an exogenous oroverexpressed polynucleotide comprising a nucleic acid encoding acoenzyme-A synthetase, which converts branched-chain fatty acid tobranched acyl-CoA. Examples of coenzyme-A synthetases include, but arenot limited to, the coenzyme-A synthetase from Leishmania braziliensis(GenBank Accession No. XP_(—)001561614), and the coenzyme-A synthetasefrom Escherichia coli (GenBank Accession No. YP_(—)541006). Optionally,the cell comprises exogenous or overexpressed polynucleotide(s)comprising a nucleic acid sequence encoding an elongase to increase thelength of the carbon backbone. Elongases are enzyme complexes thatexhibit 3-ketoacyl-CoA synthase, 3-ketoacyl-CoA reductase,3-hydroxyacyl-CoA dehydratase, and enoyl-CoA reductase activities, andgenerally utilize malonyl-CoA as an extension unit for extending thecarbon chain. When a methyl-malonyl CoA is used as an extension unit bythe enzyme complex, additional methyl branches are introduced at evencarbon positions. Exemplary elongases include, but are not limited to,elongases comprising the one or more of the following subunits:Saccharomyces cerevisiae 3-ketoacyl-CoA synthase (GenBank Accession No.NP_(—)013476), 3-ketoacyl-CoA reductase (GenBank Accession No.NP_(—)009717), 3-hydroxyacyl-CoA dehydratase (GenBank Accession No.NP_(—)012438) and enoyl-CoA reductase (GenBank Accession No.NP_(—)010269); and Arabidopsis thaliana col 3-ketoacyl-CoA synthase(GenBank Accession No. NP_(—)849861), 3-ketoacyl-CoA reductase (GenBankAccession No. NP_(—)564905), 3-hydroxyacyl-CoA dehydratase (GenBankAccession No. NP_(—)193180), and enoyl-CoA reductase (GenBank AccessionNo. NP_(—)191096).

Any suitable cell or organism, such as, for example, bacterial cells andother prokaryotic cells, and yeast cells, can be used in the context ofthe invention. In one aspect, the invention relates to cells, such asEscherichia cells (e.g., E. coli), which naturally produce Type II fattyacid synthase and/or do not naturally produce scattered branched-chainfatty acid (i.e., branched-chain fatty acid comprising a methyl branchon one or more even numbered carbons). These cells are engineered toproduce the branched-chain fatty acids as described herein.Alternatively, the cell naturally produces branched-chain fatty acid andis modified as described herein to produce higher levels ofbranched-chain fatty acid (or different proportions of different typesof branched-chain fatty acid) compared to an unmodified cell. In certainembodiments, fatty acid is manufactured using bacteria known to make themethylmalonyl-CoA precursor, such as Streptomyces, Mycobacterium orCorynebacterium. These bacteria are, in one aspect, engineered toproduce (i) an acyl transferase to increase carbon flux tomethylmalonyl-ACP that is incorporated in the fatty acid synthesispathway and/or (ii) a thioesterase to control the chain length.

Exemplary bacteria that are suitable for use in the invention include,but are not limited to, Spirochaeta aurantia, Spirochaeta littoralis,Pseudomonas maltophilia, Pseudomonas putrefaciens, Xanthomonascampestris, Legionella anisa, Moraxella catarrhalis, Thermus aquaticus,Flavobacterium aquatile, Bacteroides asaccharolyticus, Bacteroidesfragilis, Succinimonas amylolytica, Desulfovibrio africanus, Micrococcusagilis, Stomatococcus mucilaginosus, Planococcus citreus, Marinococcusalbusb, Staphylococcus aureus, Peptostreptococcus anaerobius,Ruminococcus albus, Sarcina lutea, Sporolactobacillus inulinus,Clostridium thermocellum, Sporosarcina ureae, Desulfotomaculumnigrificans, Listeria monocytogenes, Brochothrix thermosphacta,Renibacterium salmoninarum, Kurthia zopfii, Corynebacterium aquaticum,Arthrobacter radiotolerans, Brevibacterium fermentans, Propionibacteriumacidipropionici, Eubacterium lentum, Cytophaga aquatilis,Sphingobacteriuma multivorumb, Capnocytophaga gingivalis, Sporocytophagamyxococcoides, Flexibacter elegans, Myxococcus coralloides, Archangiumgephyra, Stigmatella aurantiaca, Oerskovia turbata, Escherichia coli,Bacillus subtilis, Salmonella typhimurium, Corynebacterium glutamicum,Streptomyces coelicolor, Streptomyces lividans, Clostridium thermocellumand Saccharomonospora viridis.

In one aspect, the fatty acid produced by the inventive cell comprisesabout 80% to about 100% (wt.) (e.g., about 85%, about 90%, or about 95%)linear and branched-chain fatty acid. Of the linear and branched-chainfatty acids produced by the cell, approximately 1% to approximately 95%or more (e.g., 5%, 10%, 15%, 20%, 30%, 50%, 60%, 75%, 85%, or 100%) isbranched-chain fatty acid comprising a methyl group on one or more evencarbons. In some embodiments, the cell does not produce, or producesonly trace amounts of, fatty acid comprising methyl branching on oddnumbered carbons. By “trace amount” is meant less than 1% of the totalfatty acid content produced by the cell. Alternatively or in addition,in one aspect, the mixture of fatty acids produced by the cell comprisesno more than 50% end-terminal-branched fatty acid (i.e., fatty acidsthat contain branching on a carbon atom that is within 40% of thenon-functionalized terminus of the longest carbon chain). Optionally,the inventive cell is modified to preferentially produce branched-chainfatty acid with desired chain lengths, e.g., about six to about 18carbons or more in the carbon backbone (not including the methylbranch(es)). In some embodiments, the host cell preferentially generateslong chain fatty acid, medium-length chain fatty acid, short chain fattyacid, or a desired combination fatty acids (e.g., 60%, 70%, 80%, 85%,90%, 95% or more of the branched-chain fatty acid produced by the cellcomprises the desired number of carbons). In addition, in certainembodiments, the engineered cells tolerate large amounts ofbranched-chain fatty acid in the growth medium, plasma membrane, orlipid droplets, and/or produce branched-chain fatty acid moreeconomically than an unmodified cell by, e.g., using a less expensivefeedstock, requiring less fermentation time, and the like.

The polynucleotide(s) encoding one or more polypeptides that catalyzethe reaction(s) for producing branched-chain fatty acid may be derivedfrom any source. Depending on the embodiment of the invention, thepolynucleotide is isolated from a natural source such as bacteria,algae, fungi, plants, or animals; produced via a semi-synthetic route(e.g., the nucleic acid sequence of a polynucleotide is codon-optimizedfor expression in a particular host cell, such as E. coli); orsynthesized de novo. In certain embodiments, it is advantageous toselect an enzyme from a particular source based on, e.g., the substratespecificity of the enzyme, the type of branched-chain fatty acidproduced by the source, or the level of enzyme activity in a given hostcell. In one aspect of the invention, the enzyme and correspondingpolynucleotide are naturally found in the host cell and overexpressionof the polynucleotide is desired. In this regard, in some instances,additional copies of the polynucleotide are introduced in the host cellto increase the amount of enzyme available for fatty acid production.Overexpression of a native polynucleotide also is achieved byupregulating endogenous promoter activity, or operably linking thepolynucleotide to a more robust promoter. Exogenous enzymes and theircorresponding polynucleotides also are suitable for use in the contextof the invention, and the features of the biosynthesis pathway or endproduct can be tailored depending on the particular enzyme used. Ifdesired, the polynucleotide(s) is isolated or derived from thebranched-chain fatty acid-producing organisms described herein.

In certain embodiments, the cell produces an analog or variant of apolypeptide described herein. Amino acid sequence variants of thepolypeptide include substitution, insertion, or deletion variants, andvariants may be substantially homologous or substantially identical tothe unmodified polypeptides as set out above. In certain embodiments,the variants retain at least some of the biological activity, e.g.,catalytic activity, of the polypeptide. Other variants include variantsof the polypeptide that retain at least about 50%, preferably at leastabout 75%, more preferably at least about 90%, of the biologicalactivity.

Substitution variants typically exchange one amino acid for another atone or more sites within the protein. Substitutions of this kind can beconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions include, for example, thechanges of: alanine to serine; arginine to lysine; asparagine toglutamine; aspartate to glutamate; cysteine to serine; glutamine toasparagine; glutamate to aspartate; isoleucine to leucine or valine;leucine to valine or isoleucine; lysine to arginine; methionine toleucine or isoleucine; phenylalanine to tyrosine, leucine or methionine;serine to threonine; threonine to serine; tryptophan to tyrosine;tyrosine to tryptophan or phenylalanine; and valine to isoleucine orleucine.

In some instances, the recombinant cell comprises an analog or variantof the exogenous or overexpressed polynucleotide(s) described herein.Nucleic acid sequence variants include one or more substitutions,insertions, or deletions, and variants may be substantially homologousor substantially identical to the unmodified polynucleotide.Polynucleotide variants or analogs encode mutant enzymes having at leastpartial activity of the unmodified enzyme. Alternatively, polynucleotidevariants or analogs encode the same amino acid sequence as theunmodified polynucleotide. Codon-optimized sequences, for example,generally encode the same amino acid sequence as the parent/nativesequence but contain codons that are preferentially expressed in aparticular host organism.

A polypeptide or polynucleotide “derived from” an organism contains oneor more modifications to the native amino acid sequence or nucleotidesequence and exhibits similar, if not better, activity compared to thenative enzyme (e.g., at least 70%, at least 80%, at least 90%, at least95%, at least 100%, or at least 110% the level of activity of the nativeenzyme). For example, enzyme activity is improved in some contexts bydirected evolution of a parent/native sequence. Additionally oralternatively, an enzyme coding sequence is mutated to achieve feedbackresistance. Thus, in one or more embodiments of the invention, thepolypeptide encoded by the exogenous polynucleotide is feedbackresistant and/or is modified to alter the activity of the native enzyme.A polynucleotide “derived from” a reference polynucleotide encompasses,but is not limited to, a polynucleotide comprising a nucleic acidsequence that has been codon-optimized for expression in a desired hostcell.

The cell of the invention may comprise any combination ofpolynucleotides described herein to produce branched-chain fatty acidcomprising a methyl branch on one or more even number carbons. Forexample, the invention provides a cell comprising (i) an exogenous oroverexpressed polynucleotide comprising a nucleic acid sequence encodingan acyl transferase lacking polyketide synthesis activity, and (ii) anexogenous or overexpressed polynucleotide comprising a nucleic acidsequence encoding a propionyl-CoA carboxylase and/or an exogenous oroverexpressed polynucleotide comprising a nucleic acid sequence encodinga methylmalonyl-CoA mutase, wherein the polynucleotide(s) are expressedand the cell produces more branched-chain fatty acid comprising a methylon one or more even number carbons than an otherwise similar cell thatdoes not comprise the polynucleotide(s). Recombinant cells can beproduced in any suitable manner to establish an expression vector withinthe cell. The expression vector can include the exogenous polynucleotideoperably linked to expression elements, such as, for example, promoters,enhancers, ribosome binding sites, operators and activating sequences.Such expression elements may be regulatable, for example, inducible (viathe addition of an inducer). Alternatively or in addition, theexpression vector can include additional copies of a polynucleotideencoding a native gene product operably linked to expression elements.Representative examples of useful promoters include, but are not limitedto: the LTR (long terminal 35 repeat from a retrovirus) or SV40promoter, the E. coli lac, tet, or trp promoter, the phage Lambda P_(L)promoter, and other promoters known to control expression of genes inprokaryotic or eukaryotic cells or their viruses. In one aspect, theexpression vector also includes appropriate sequences for amplifyingexpression. The expression vector can comprise elements to facilitateincorporation of polynucleotides into the cellular genome. Introductionof the expression vector or other polynucleotides into cells can beperformed using any suitable method, such as, for example,transformation, electroporation, microinjection, microprojectilebombardment, calcium phosphate precipitation, modified calcium phosphateprecipitation, cationic lipid treatment, photoporation, fusionmethodologies, receptor mediated transfer, or polybrene precipitation.Alternatively, the expression vector or other polynucleotides can beintroduced by infection with a viral vector, by conjugation, bytransduction, or by other any other suitable method.

Cells, such as bacterial cells, containing the polynucleotides encodingthe proteins described herein can be cultured under conditionsappropriate for growth of the cells and expression of thepolynucleotides. Cells expressing the protein can be identified by anysuitable methods, such as, for example, by PCR screening, screening bySouthern blot analysis, or screening for the expression of the protein.In certain embodiments, cells that contain the polynucleotide(s) can beselected by including a selectable marker in the DNA construct, withsubsequent culturing of cells containing a selectable marker gene, underconditions appropriate for survival of only those cells that express theselectable marker gene. The introduced DNA construct can be furtheramplified by culturing genetically modified cells under appropriateconditions (e.g., culturing genetically modified cells containing anamplifiable marker gene in the presence of a concentration of a drug atwhich only cells containing multiple copies of the amplifiable markergene can survive). Cells that contain and express polynucleotidesencoding the exogenous proteins can be referred to herein as geneticallymodified cells. Bacterial cells that contain and express polynucleotidesencoding the exogenous protein can be referred to as geneticallymodified bacterial cells.

Exemplary cells of the invention include E. coli BW25113 comprisingpTrcHisA mmat and pZA31-accA1-pccB, which was deposited with AmericanType Culture Collection (ATCC), 10801 University Blvd., Manassas, Va.,on Dec. 14, 2010, under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure (“Budapest Treaty”), and assigned DepositAccession No. [XXX] on [DATE], and E. coli BL21 Star (DE3) comprisingpTrcHisA Ec sbm So ce epi and pZA31 mmat which was deposited withAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va., on Dec. 14, 2010, under the provisions of the BudapestTreaty for the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure (“Budapest Treaty”),and assigned Deposit Accession No. [XXX] on [DATE]. The invention alsoincludes variants or progeny of the cells described herein that retainthe phenotypic characteristics of the recombinant microbe. Asubstantially pure monoculture of the cell described herein (i.e., aculture comprising at least 80% or at least 90% of a desired cell) alsois provided.

Any cell culture conditions appropriate for growing a host cell andsynthesizing branched-chain fatty acid is suitable for use in theinventive method. Addition of fatty acid synthesis intermediates,precursors, and/or co-factors for the enzymes associated withbranched-chain fatty acid synthesis to the culture is contemplatedherein. In certain embodiments, the genetically modified cells (such asgenetically modified bacterial cells) have an optimal temperature forgrowth, such as, for example, a lower temperature than normallyencountered for growth and/or fermentation. For example, in certainembodiments, incorporation of branched-chain fatty acids into themembrane may increase membrane fluidity, a property normally associatedwith low growth temperatures. In addition, in certain embodiments, cellsof the invention may exhibit a decline in growth at higher temperaturesas compared to normal growth and/or fermentation temperatures astypically found in cells of the type.

The inventive method optionally comprises extracting branched-chainfatty acid from the culture. Fatty acids can be extracted from theculture medium and measured using any suitable manner. Suitableextraction methods include, for example, methods as described in: Blighet al., A rapid method for total lipid extraction and purification, Can.J. Biochem. Physiol. 37:911-917 (1959). In certain embodiments,production of fatty acids in the culture supernatant or in the membranefraction of recombinant cells can be measured. In this embodiment,cultures are prepared in the standard manner, although nutrients (e.g.,2-methylbutyrate, isoleucine) that may provide a boost in substratesupply can be added to the culture. Cells are harvested bycentrifugation, acidified with hydrochloric or perchloric acid, andextracted with chloroform and methanol, with the fatty acids enteringthe organic layer. The fatty acids are converted to methyl esters, usingmethanol at 100° C. The methyl esters are separated by gaschromatography (GC) and compared with known standards of fatty acids(purchased from Larodan or Sigma). Confirmation of chemical identity iscarried out by combined GC/mass spec, with further mass spec analysis offragmented material carried out if necessary.

In one embodiment, the cell utilizes the branched-chain fatty acid as aprecursor to make one or more other products. Products biosynthesized(i.e., derived) from branched-chain fatty acid include, but are notlimited to, phospholipids, triglycerides, alkanes, olefins, wax esters,fatty alcohols, and fatty aldehydes. Some host cells naturally generateone or more products derived from branched-chain fatty acid; other hostcells are genetically engineered to convert branched-chain fatty acidto, e.g., an alkane, olefin, wax ester, fatty alcohol, phospholipid,triglyceride, and/or fatty aldehyde. Organisms and genetic modificationsthereof to synthesize products derived from branched-chain fatty acidsare further described in, e.g., International Patent Publication Nos. WO2007/136762, WO 2008/151149, and WO 2010/062480, and U.S. PatentApplication Publication US 2010/0298612, all of which are herebyincorporated by reference in their entirety. In one aspect, theinventive method comprises extracting a product derived frombranched-chain fatty acid (phospholipid, triglyceride, alkane, olefin,wax ester, fatty alcohol, and/or fatty aldehyde synthesized in the cellfrom branched-chain fatty acid) from the culture. Any extraction methodis appropriate, including the extraction methods described inInternational Patent Publication Nos. WO 2007/136762, WO 2008/151149,and WO 2010/062480, and U.S. Patent Application Publication Nos. US2010/0251601, US 20100242345, US 20100105963, and US 2010/0298612.

The inventive cell preferably produces more branched-chain fatty acidcomprising a methyl branch on one or more even number carbons than anotherwise similar cell that does not comprise the polynucleotide(s).Methods of measuring fatty acid released into the fermentation broth orculture media or liberated from cellular fractions are described herein.Branched-chain fatty acid production is not limited to fatty acidaccumulated in the culture, however, but also includes fatty acid usedas a precursor for downstream reactions yielding products derived frombranched-chain fatty acid. Thus, products derived from branched-chainfatty acid (e.g., phospholipids, triglycerides, fatty alcohols, olefins,wax esters, fatty aldehydes, and alkanes) are, in some embodiments,surrogates for measuring branched-chain fatty acid production in a hostcell. Methods of measuring fatty acid content in phospholipid in thecell membrane are described herein. Similarly, measurement ofdegradation products of branched-chain fatty acids also is instructiveas to the amount of branched-chain fatty acid is produced in a hostcell. Depending on the particular embodiment of the invention, theinventive cell produces at least 3%, at least 5%, at least 10%, at least20%, at least 25%, or at least 50% more branched-chain fatty acid thanan otherwise similar cell that does not comprise the polynucleotide(s).

The invention further provides a composition comprising thebranched-chain fatty acids described herein. For example, the inventionprovides a composition comprising a branched-chain fatty acid comprisingbetween 10-18 carbons in the carbon backbone, such as fatty acidscomprising between 10 and 16 carbons (e.g., fatty acids comprising 10,11, 12, 13, 14, 15, or 16 carbons), with branching on one or more evennumbered carbons (e.g., C2, C4, C6, C8, C10, C12, C14, and/or C16). Acomposition comprising longer-chain fatty acid also is provided, such asa composition comprising between 19 and 22 carbons in the longest carbonchain. A composition comprising a combination of any of the fatty acidsdescribed herein also is provided (e.g., a composition comprising fattyacids of varying lengths and/or branch locations along the carbonbackbone).

The following examples further describe and demonstrate embodimentswithin the scope of the invention. The examples are given solely for thepurpose of illustration and are not to be construed as limitations ofthe invention, as many variations thereof are possible without departingfrom the spirit and scope of the invention.

Example 1 Construction of Methylmalonyl-CoA Mutase Expression Vector

There are numerous genes annotated to encode the two subunits ofmethylmalonyl-CoA mutase. Janibacter sp. HTCC2649 encodes two suchgenes. Synthetic versions of these genes were prepared, with the codonusage altered to match that used by many E. coli genes (i.e., the codingsequence was codon-optimized for expression in E. coli). By analogy toother methylmalonyl-CoA mutase genes, these synthetic genes were namedmutA (SEQ ID NO: 1) and mutB (SEQ ID NO: 2), corresponding to the MutA(SEQ ID NO: 3) and MutB (SEQ ID NO: 4) protein subunits. In thesynthetic DNA, an extra three base pairs were added (encoding an alanineresidue immediately after the initiation methionine) in mutA tofacilitate introduction of an NcoI site. An XhoI restriction site wasalso placed after the coding sequence of mutB for insertion into thepBAD vector (Invitrogen). The NcoI/XhoI fragment was cloned into pBAD.

Example 2 Construction of Methylmalonyl-CoA Epimerase Expression Vector

There are numerous genes annotated to encode methylmalonyl-CoA mutase.One such gene is from Streptomyces sviceus. A synthetic gene can beconstructed (SEQ ID NO: 5) using codon usage similar to E. coli genesand with EcoRI and Hind III sites flanking the coding region. An E. coliShine-Dalgarno sequence can be added between the EcoRI site and theinitiation codon for the epimerase gene. The predicted protein productis the same as the predicted protein product from the S. sviceus gene(SEQ ID NO: 6). The epimerase gene can be cloned into the pBAD-mutABconstruct using the EcoRI and Hind III restriction sites (downstream ofmutB) to form the pBAD-mutAB-epimerase gene plasmid. E. coli culturescan be grown at 27° C. after induction with arabinose and supplementedwith hydroxycobalamin to achieve expression of functionalmethylmalonyl-CoA mutase and branched-chain fatty acid production.

Example 3 Construction of Propionyl-CoA Carboxylase Expression Vector

Nucleotide sequences (SEQ ID NO: 7 and SEQ ID NO: 8) encoding the twopropionyl-CoA carboxylase subunits AccA1 (GenBank Accession NO.AF113603.1; SEQ ID NO: 9) and PccB (GenBank Accession No. AF113605.1;SEQ ID NO: 10)), respectively, from the Streptomyces coelicolor A3(2)propionyl-CoA carboxylase (Rodriguez E., Gramajo H., Microbiology. 1999November; 145:3109-19), were codon-optimized for E. coli expression. Agene construct for expressing propionyl-CoA carboxylase was constructedwith the following elements sequentially 1) P_(Llac0-1) promoter andoperator plus T7 gene10 ribosomal binding site (SEQ ID NO: 11); 2)optimized accA1 (SEQ ID NO: 12); 3) three restriction site sequencesincluding BglII, NotI and XbaI and a T7 gene10 ribosome binding site(SEQ ID NO: 13); and 4) codon-optimized pccB (SEQ ID NO: 14). Thesynthesized DNA fragments were cloned into the XhoI and PstI sites ofexpression vector pZA31-MCS (Expressys, Ruelzheim, Germany), resultingin plasmid pZA31-accA1-pccB (SEQ ID NO: 15).

Example 4 Construction of Propionyl-CoA Synthetase Expression Vector

The Salmonella enterica propionyl-CoA synthetase gene, prpE, wasamplified using PCR and the primers set forth in SEQ ID NO: 16 and SEQID NO: 17, and placed behind a Shine-Dalgarno sequence in the plasmidpZA31-accA1-pccB (SEQ ID NO: 15) using the restriction enzymes PstI andBamHI. Enhanced propionyl-CoA synthetase production is expected toincrease synthetic flux to propionyl-CoA.

Example 5 Reduction of Propionylation of Propionyl-CoA Synthetase

In S. enterica, propionyl-CoA synthetase is subject to inhibition bypropionylation at lysine 592 when propionyl-CoA levels accumulate.(Garrity et al, J. Biol. Chem., Vol. 282, Issue 41, 30239-30245, Oct.12, 2007). Similar enzyme modulation may occur in other species,although the position of the modified lysine may be different. Severalstrategies to overcome this inhibition will be tested and compared.First, the propionyl-CoA synthetase gene will be mutated to change thecoding capacity from lysine (at the site of propionylation) to arginineor other amino acids to prevent propionylation. Second, a source ofresveratrol or other sirtuin activators will be introduced into theculture medium to activate sirtuin to depropionylate PrpE. Third, theendogenous N-acetyltransferase enzyme responsible for the propionylationreaction will be knocked out. For example, if working with S. enterica,pat could be deleted. As another example, if working with B. subtilis,acuA could be deleted. Fourth, the flux of propionyl-CoA into fatty acidsynthesis will be increased by increasing propionyl-CoA carboxylaseactivity to keep free propionyl-CoA levels down. Fifth, the sirtuinactivity will be increased, thus increasing deacetylation ofpropionyl-CoA carboxylase. For example, the S. enterica cobB expressioncould be increased.

Example 6 Creation of an Expression Vector Comprising the CodingSequence of the MMAT (Methylmalonyl-CoA Acyl Transferase) Domain fromMycobacterium Mycocerosic Acid Synthase (MAS).

Mycobacterium MAS is a multifunctional protein that catalyzes thesynthesis of mycocerosic acid and that contains a domain with MMATactivity. The MMAT domain (amino acids 508-890) (SEQ ID NO: 18) of MASfrom Mycobacterium bovis BCG (YP_(—)979046) (SEQ ID NO: 19) was codonoptimized for E. coli expression (SEQ ID NO: 20). The optimized sequencewas synthesized and cloned into vector pTrcHisA (Invitrogen) between theBamHI and HindIII sites. The resulting construct fused the MMAT domainwith the His tag leader peptide encoded by the vector. The expressionvector was introduced into a recombinant E. coli host that producesmethylmalonyl-CoA. MMAT activity catalyzes the formation ofmethylmalonyl-ACP, which subsequently can be incorporated into the typeII fatty acid synthesis pathway to form methyl branches at evenpositions of the fatty acid chain.

Example 7 Method for Detecting Acyl-CoA

This example describes an exemplary method for detecting and quantifyingan acyl-CoA (e.g., methylmalonyl-CoA) in a sample, such as a sample ofrecombinant host cells producing branched-chain fatty acid.

A stable, labeled (deuterium) internal standard-containing master mixwas prepared comprising d₃-3-hydroxymethylglutaryl-CoA (200 μl of 50μg/ml stock in 10 ml of 15% trichloroacetic acid). An aliquot (500 μl)of the master mix was added to a 2 ml tube. Silicone oil (AR200; Sigmacatalog number 85419; 800 μl) was layered onto the master mix. An E.coli culture (800 μl) was layered gently on top of the silicone oil, andthe resulting sample was subjected to centrifugation at 20,000×g forfive minutes at 4° C. in an Eppendorf 5417 C centrifuge. A portion (300μl) of the master mix-containing layer was transferred to an empty tubeand frozen on dry ice for 30 minutes.

The acyl-CoA content of samples was determined using HPLC/MS/MS.Individual coenzyme-A standards (propionyl-CoA, methylmalonyl-CoA,succinyl-CoA, malonyl-CoA, isobutyryl-CoA, isovaleryl-CoA, andacetyl-CoA) were purchased from Sigma Chemical Company (St. Louis, Mo.)and prepared as 500 μg/ml stocks in methanol. The analytes were pooled,and standards with all of the analytes were prepared by dilution with15% trichloroacetic acid. Standards for regression were prepared bytransferring 500 μl of the working standards to an autosampler vialcontaining 10 μL of the 50 μg/ml internal standard. Sample peak areas(or heights) were normalized to the stable-labeled internal standard(d₃-3-hydroxymethylglutaryl-CoA, Cayman Chemical Co.). Samples wereassayed by HPLC/MS/MS on a Sciex API5000 mass spectrometer in positiveion Turbo Ion Spray. Separation was carried out by reversed-phase highperformance liquid chromatography using a Phenomenex Onyx Monolithic C18column (2×50 mm) and mobile phases of (1) 5 mM ammonium acetate, 5 mMdimethylbutylamine, 6.5 mM acetic acid and (2) acetonitrile with 0.1%formic acid, with the gradient set forth in Table C.

TABLE C Mobile Mobile Phase A Phase B Time (%) (%)   0 min 97.5 2.5 1.0min 97.5 2.5 2.5 min 91.0 9.0 5.5 min 45 55 6.0 min 45 55 6.1 min 97.52.5 7.5 min — — 9.5 min End Run

The conditions on the mass spectrometer were: DP 160, CUR 30, GS1 65,GS2 65, IS 4500, CAD 7, TEMP 650 C. The transitions set forth in Table Dwere used for the multiple reaction monitoring (MRM).

TABLE D Precursor Product Collision Compound Ion* Ion* Energy CXPn-Propionyl-CoA 824.3 317.2 41 32 Methylmalonyl-CoA 868.1 317.1 42 31Succinyl-CoA 868.2 361.1 49 38 Malonyl-CoA 854.2 347.2 41 36Isobutyryl-CoA 838.3 345.2 45 34 Isovaleryl-CoA 852.2 345.2 45 34Acetyl-CoA 810.3 303.2 43 30 d3-3-Hydroxymethylglutaryl- 915.2 408.2 4913 CoA *Energy (Volts) for MS/MS analysis

Example 8 Analysis of Fatty Acids Produced by Host Cells

This example illustrates a method of analyzing branched-chain fattyacids produced by cells (e.g., recombinant microbes).

Cell cultures (approximately 1.5 ml) were frozen in 2.0 ml glass vialsand stored at −20° C. until ready for processing. Samples were chilledon dry ice for 30 minutes and lyophilized overnight (−16 hours) untildry. A 10 μl aliquot of internal standard (glyceryl trinonadecanoate(Sigma catalog number T4632-1G)) was added to each vial, followed by 400μL of 0.5 N NaOH (in methanol). The vial was capped and vortexed for 10seconds. Samples were incubated at 65°C. for 30-50 minutes. Samples werethen removed from the incubator, and 500 μl of boron trifluoride reagent(Aldrich catalog number B1252) was added. The samples were vortexedagain for 10 seconds, incubated at 65° C. for 10-15 minutes, and cooledto room temperature (approximately 20 minutes). Hexane (350 μl) wasadded, and the samples were again vortexed for 10 seconds. If the phasesdid not separate, 50-100 μl of saturated salt solution (5 g NaCl to 5 mlwater) was added, and the sample was vortexed for 10 seconds. At least100 μl of the top hexane layer was placed into the gas chromatographyvial. The vial was capped and stored at 4° C. until analyzed by gaschromatography.

Gas chromatography was performed as described in Table E below. Abacterial acid methyl ester standard (Sigma catalog number 47080-U) anda fatty acid methyl ester standard (Sigma catalog number 47885-U) wereused to identify peaks in samples. A sample check standard usingglyceryl tripalmitate (Sigma catalog number T5888-1G) was used toconfirm esterification of samples. A blank standard (internal standardonly) was used to assess background noise.

TABLE E Gas Chromatograph HP 5890 GC Series II Detector FID 360° C. 40ml/min Hydrogen, 400 ml/min Air Carrier Gas Helium Quantitative GCChemstation A.09.03. (Agilent) Program Column VF-5 ms 15 M × 0.150 mm ×0.15 μm Varian catalog number CP9035 Injection Liner Gooseneck (withglass wool packing) Injector HP 7673 Injection Syringe 10 μL InjectionMode Split 25:1 Injection volume 4 μL (Plunger Speed = fast; 5 samplepumps) Pre Injection Solvent 2 samples Washes Post Injection 3 for bothacetone and hexane Solvent Washes Injector Temperature 325° C. TotalProgram Time 16 minutes Initial Initial Final Final Temp. Time Rate TempTime (° C.) (min) (° C./min) (° C.) (min) Thermal Program 90 0.75 20.0325 1.0 25.0 350 2.5

Example 9 Construction of Expression Vectors Comprising S. CinnamonensismutA and mutB and S. sviceus epi.

A synthetic DNA construct was generated comprising Streptomycescinnamonensis mutA (SEQ ID NO: 24) (GenBank Accession No. AAA03040.1),S. cinnamonensis mutB (SEQ ID NO: 25) (GenBank Accession No.AAA03041.1), and a Streptomyces sviceus ATCC 29083 methylmalonyl-CoAepimerase gene (SEQ ID NO: 26) (GenBank Accession No. ZP_(—)06919825.1).The genes were codon-optimized for expression in E. coli. An EcoRIrestriction site was placed on the 5′ end, and a BamHI site was placedon the 3′ end of the synthesized gene construct. These sites weresubsequently used for cloning into a pZA31 vector (Expressys, Ruelzheim,Germany). A ribosome binding sequence and spacer was placed before themutA and epimerase gene start codons (SEQ ID NO: 27). The plasmid wasdesignated pZA31 mutAB Ss epi.

Example 10 Construction of Expression Vectors Comprising Sbm andmalE/sbm Polynucleotides

Sleeping beauty mutase (Sbm) (also known as methylmalonyl-CoA mutase(MCM)) is an enzyme that catalyzes the rearrangement of succinyl-CoA toL-methylmalonyl-CoA. The enzyme is vitamin B12 (cobalamin) dependent.Methylmalonyl-CoA is a building block for scattered branch-chain fattyacids (sBCFA) (i.e., branched-chain fatty acid comprising a methylbranch on one or more even number carbons of the fatty acid backbone).Plasmids comprising a polynucleotide encoding Sbm were generated tointroduce multiple copies of the Sbm coding sequence, downstream of aregulatable promoter, into E. coli host cells.

A polynucleotide was synthesized based on the sequence of E. coli sbm(SEQ ID NO: 28) (GenBank Accession No. NP_(—)417392.1) from E. colistrain MG1655. The nucleic acid sequence was codon-optimized to matchthe pattern of highly expressed E. coli genes while maintaining thenative amino acid sequence of the enzyme. The generated nucleic acidsequence is set forth in SEQ ID NO: 29. A BamHI and an XbaI site wereadded at the 5′ end of the synthetic Sbm coding sequence with thesequence GGATCCATGTCTAGA (SEQ ID NO: 49) adjacent to the ATG translationinitiation sequence. A SacI restriction site sequence was added to the3′ end of the synthetic Sbm coding sequence. The gene was synthesized,cloned into a pUC57 vector, and sequenced (GenScript, Piscataway, N.J.).The synthetic sbm was then released from pUC57 by restriction enzymesBamHI and Sad, and sub-cloned into plasmid pTrcHisA (Invitrogen,Carlsbad, Calif.) in frame with the poly-histidine sequence (GenScript,Piscataway, N.J.). The plasmid was designated pTrcHisA Ec sbm. Thesequence was confirmed by sequencing (GenScript, Piscataway, N.J.). Therecombinant protein encoded by the sequence contained a poly-histidinesequence(Met-Gly-Gly-Ser-His-His-His-His-His-His-Gly-Met-Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg-Thr-Asp-Asp-Asp-Asp-Lys-Asp-Arg-Trp-Gly-Ser(SEQ ID NO: 50)) and a full-length native Sbm amino acid sequence.

A recombinant methylmalonyl-CoA mutase has been reported to be insolublein E. coli (Korotkova, N., and M. E. Lidstrom. J. Biological Chemistry279: 13652-8 (2004)). Translation fusion with maltose-binding protein(MBP, encoded by malE) prevents aggregation of recombinant proteins(Kapust, R. B., and D. S. Waugh. Protein Science 8: 1668-74 (1999)). Arecombinant construct was generated by inserting malE upstream of sbm.The malE polynucleotide was synthesized based on the sequence of maltosebinding protein (E. coli MG1655 GenBank NC_(—)000913.2 (GenScript,Piscataway, N.J.)). A BamHI site was placed adjacent to the translationinitiation codon of malE, and an XbaI site was placed immediately 5′ tothe stop codon of the malE sequence (SEQ ID NO: 30). Also, onenucleotide was changed (T438 to C438) to remove a restriction siterecognition sequence for BglII.

The MalE coding sequence (SEQ ID NO: 30) was first synthesized andcloned into a pUC57 plasmid. After confirming its sequence, the malEpolynucleotide was released using restriction enzymes BamHI and XbaI.The released malE was then re-cloned into plasmid pTrcHisA Ec sbm atBamHI and XbaI sites (GenScript, Piscataway, N.J.). The resultingplasmid was designated pTrcHisA Ec malE Ec sbm. The recombinant proteinencoded by pTrcHisA Ec malE Ec sbm contains three peptides: thepoly-histidine tag, full-length MBP, and full-length Sbm.

Example 11 Construction of a Recombinant Expression Vector Comprising aPolynucleotide Encoding the Methylmalonyl-CoA Acyl Transferase (MMAT)Domain from Mycobacterium Mycocerosic Acid Synthase (MAS).

Mycobacterium MAS is a multifunctional protein containing MMAT activitythat catalyzes the synthesis of mycocerosic acid. The nucleic acidsequence encoding the MMAT domain (amino acids 508-890) (SEQ ID NO: 18)of MAS from Mycobacterium bovis BCG (GenBank Accession No. YP_(—)979046)(SEQ ID NO: 19) was codon-optimized for E. coli expression (SEQ ID NO:20). The optimized sequence, designated “mmat,” was synthesized andcloned into vector pTrcHisA (Invitrogen) between the BamHI and HindIIIsites. The resulting construct fused the MMAT domain with thepoly-histidine tag encoded by the vector. The expression vector(pTrcHisA mmat) was introduced into a recombinant E. coli host thatproduces methylmalonyl-CoA. MMAT activity catalyzes the formation ofmethylmalonyl-ACP, which is incorporated by Type II fatty acid synthaseinto fatty acid, forming methyl branches at even positions of the fattyacid chain.

An expression vector encoding Mycobacterium bovis BCG fused to apoly-histidine tag also was generated. The pTrcHisA mmat plasmid DNAdescribed above was amplified by PCR using oligonucleotides synthesizedto include 5′-KpnI (SEQ ID NO: 31) and 3′-HindIII restriction sites (SEQID NO: 32) (Integrated DNA Technologies, Inc., Coralville, Iowa). PCRwas run on samples having 1 μl (2 ng) pTrcHisA mmat DNA, 1.5 μl of a 10μM stock of each primer, 5 μl of 10× Pfx reaction mix (InvitrogenCarlsbad, Calif.), 0.5 μl of Pfx DNA polymerase (1.25 units), and 41 μlof water. PCR conditions were as follows: the samples were initiallyincubated at 95° C. for three minutes, followed by 30 cycles at 95° C.for 30 seconds (strand separation), 58° C. for 30 seconds (primerannealing), and 68° C. primer extension for 1.5 minutes. Following thecycles, the samples were incubated for 10 minutes at 68° C., and thesamples were then held at 4° C.

The PCR products were purified using a QIAquick® PCR Purification Kit(Qiagen), digested with restriction enzymes KpnI and HindIII and ligated(Fast-Link Epicentre Biotechnologies, Madison, Wis.) withKpnI/HindIII-digested pZA31MCS (Expressys, Ruelzheim, Germany). Theligation mix was used to transform E. coli DHSα™ (Invitrogen Carlsbad,Calif.). Isolated colonies were screened by PCR using a sterile pipettetip stab as an inoculum into a reaction tube containing only water,followed by addition of the remaining PCR reaction cocktail (AccuPrime™SuperMixII, Invitrogen Carlsbad, Calif.) and primers as described above.

Recombinant plasmids were isolated and purified using the QIAPrep® SpinMiniprep Kit (Qiagen) and characterized by restriction enzyme digestion(DraI, KpnI and HindIII from New England Biolabs, Beverly, Mass.). Theplasmids were subsequently used to transform BW25113 (E. coli GeneticsStock Center, New Haven, Conn.) made competent using the calciumchloride method. Transformants were selected on Luria agar platescontaining 34 μg/ml chloramphenicol. Plasmid DNA was isolated andpurified using the QIAfilter™ Plasmid Midi Kit (Qiagen). DNA sequencingconfirmed that the insert was mmat (SEQ ID NO: 34). The resultingplasmid incorporating a poly-histidine tag was designated pZA31 mmat.

Example 12 Method of Generating a Recombinant Host Cell Comprising anExogenous Polynucleotide Encoding a Propionyl-CoA Carboxylase and anExogenous Polynucleotide Encoding a Methylmalonyl-CoA Acyl Transferase(MMAT) Domain from Mycobacterium Mycocerosic Acid Synthase (MAS).

This example describes an exemplary method for making a cell comprisingan exogenous polynucleotide comprising a nucleic acid sequence encodinga polypeptide that catalyzes the conversion of propionyl-CoA tomethylmalonyl-CoA and an exogenous polynucleotide comprising a nucleicacid sequence encoding a polypeptide that catalyzes the conversion ofmethylmalonyl-CoA to methylmalonyl-ACP. The method entailsco-transduction of E. coli with plasmids containing a propionyl-CoAcarboxylase gene from Streptomyces coelicolor and a gene encoding a MMATdomain from Mycobacterium MAS.

E. coli BW25113 cells (E. coli Genetic Stock Center, New Haven, Conn.)were made chemically competent for plasmid DNA transformation by acalcium chloride method. Actively growing 50 ml E. coli cultures weregrown to an optical density (at 600 nm) of ˜0.4. Cultures were quicklychilled on ice, and the bacteria were recovered by centrifugation at2700×g for 10 minutes. The supernatant was discarded and pellets weregently suspended in 30 ml of an ice-cold 80 mM MgCl₂, 20 mM CaCl₂solution. Cells were again recovered by centrifugation at 2700×g for 10minutes. The supernatant was discarded and pellets were gentlyresuspended in 2 ml of an ice-cold 0.1 M CaCl₂ solution.

Cells were transformed on ice in pre-chilled 14 ml round-bottomcentrifuge tubes. Approximately 25 ng of each of pTrcHisA mmat andpZA31-accA1-pccB (described above) was incubated on ice with 100 μl ofcompetent cells for 30 minutes. The cells were heat shocked at 42° C.for 90 seconds and immediately placed on ice for two minutes. Pre-warmedSOC medium (500 μl; Invitrogen, Carlsbad, Calif.) was added and thecells allowed to recover at 37° C. with 225 rpm shaking. A portion (50μl) of the transformed cell mix was spread onto selective LB agar 100mg/ml ampicillin and 34 mg/ml chloramphenicol plates to select for cellscarrying the pTrcHisA mmat and pZA31/32-accA1-pccB plasmids. Individualcolonies were picked from each plate and streaked onto LB agar (withampicillin and chloramphenicol) to confirm the antibiotic resistancephenotype. Restriction endonuclease digestion analysis of isolatedplasmid DNA with HaeII verified the plasmid DNA pool for each strain. Asample of E. coli BW25113 comprising pTrcHisA mmat and pZA31-accA1-pccBwas deposited with American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va., on Dec. 14, 2010, under the provisionsof the Budapest Treaty for the International Recognition of the Depositof Microorganisms for the Purpose of Patent Procedure (“BudapestTreaty”), and assigned Deposit Accession No. [XXX] on [DATE].

Example 13 Construction of an Expression Vector Encoding SorangiumCellulosum So ce 56 Methylmalonyl-CoA Epimerase

A S. cellulosum methylmalonyl-CoA epimerase synthetic gene (So ce epi)was designed and synthesized (SEQ ID NO: 37). The coding sequence wascodon-optimization for expression in E. coli and modified to removerestriction sites (GenScript, Piscataway, N.J.). The nucleic acidsequence was flanked with a SacI site and a synthetic ribosome bindingsite from the pBAD vector (Invitrogen, Carlsbad, Calif.) adjacent to thetranslation initiation sequence (SEQ ID NO: 39). The synthetic gene wascloned as a SacI/PstI fragment into pTrcHisA Ec sbm and pTrcHisA Ec malEEc sbm, with the resulting plasmids designated as pTrcHisA Ec sbm So ceepi and pTrcHisA Ec malE Ec sbm So ce epi, respectively.

Example 14 Construction of an Expression Vector Encoding KribbellaFlavida DSM 17836 Methylmalonyl-CoA Epimerase

A K. flavida methylmalonyl-CoA epimerase gene (Kf epi) was designed andsynthesized (SEQ ID NO: 35). The coding sequence was optimized forexpression in E. coli and restriction sites were removed (GenScript,Piscataway, N.J.). The gene was flanked with a Sad site and a syntheticribosome binding site from the pBAD vector adjacent to the translationinitiation sequence (SEQ ID NO: 39). The synthetic gene was cloned as aSacI/PstI fragment into pTrcHisA Ec sbm and pTrcHisA Ec malE Ec sbm. Theresulting plasmids were designated pTrcHisA Ec sbm Kf epi and pTrcHisAEc malE Ec sbm Kf epi, respectively.

Example 15 Production of Host Cells Producing Branched-Chain Fatty Acid

This example describes the production of branched-chain fatty acid usinga recombinant host cell (e.g., E. coli) expressing polynucleotidesencoding a propionyl-CoA carboxylase or a methylmalonyl-CoA mutase and amethylmalonyl-CoA epimerase, in some instances in conjunction with apolynucleotide encoding an acyl transferase and/or thioesterase.

It is useful to have the capacity to tailor the fatty acid chain length.Branched fatty acids of different lengths have different physicalproperties suitable for different commercial applications. Todemonstrate the capacity to tailor the chain length of branched fattyacids, E. coli 'TesA (Cho, H., and J. E. Cronan, Jr. J. BiologicalChemistry 270: 4216-9 (1995)) was incorporated into expression vectorsdescribed above and inserted into host cells. To create a pTrc Ec 'tesAexpression vector, a truncated E. coli tesA ('tesA) cDNA (SEQ ID NO: 40)was created by PCR amplification of the E. coli tesA gene (GenBankAccession No. L06182). A 5′ primer (SEQ ID NO: 41) was designed toanneal after the 26th codon of tesA, modifying the 27th codon from analanine to a methionine and creating a NcoI restriction site. A 3′primer (SEQ ID NO: 43) incorporating a BamHI restriction site wasdesigned. PCR was performed with 50 μl of Pfu Ultra II Hotstart 2×master mix (Agilent Technologies, Santa Clara, Calif.), 1 μl of a mix ofthe two primers (10 μmoles of each), 1 μl of E. coli BW25113 genomicDNA, and 48 μl of water. PCR began with a two minute incubation at 95°C., followed by 30 cycles of 20 seconds at 95° C. for denaturation, 20seconds for annealing at 58° C., and 15 seconds at 72° C. for extension.The sample was incubated at 72° C. for three minutes and then held at 4°C. The PCR product (Ec 'tesA) was purified using a QIAquick® PCRPurification Kit (Qiagen, Valencia, Calif.). The bacterial expressionvector pTrcHisA and the 'tesA PCR product were digested with NcoI andBamHI. The digested vector and insert were ligated using Fast-Link(Epicentre Biotechnologies, Madison, Wis.). The ligation mix was thenused to transform E. coli TOP 10 cells (Invitrogen, Carlsbad, Calif.).Recombinant plasmids were isolated using a QIAPrep0 Spin Miniprep Kit(Qiagen) and characterized by gel electrophoresis of restriction digestswith HaeII. DNA sequencing confirmed that the 'tesA insert had beencloned and that the insert encoded the expected amino acid sequence (SEQID NO: 45). The resulting plasmid was designated pTrc Ec 'tesA.

To limit gene expression, the truncated E. coli 'tesA gene was subclonedinto the low-copy bacterial expression vector pZS21-MCS (Expressys,Ruelzheim, Germany). The expression vector pTrc Ec 'tesA was a templatein a PCR reaction using a 5′ primer designed to create a flanking XhoIrestriction site and include the pTrcHisA lac promoter (to replace thepZS21-MCS vector tet promoter) (SEQ ID NO: 46) and a 3′ primerincorporating a HindIII restriction site (SEQ ID NO: 47). PCR wasperformed with 50 μl of Pfu Ultra II Hotstart 2× master mix (AgilentTechnologies, Santa Clara, Calif.), 1 μl of a mix of the two primers (10μmoles of each), 1 μl of pTrc Ec 'tesA plasmid DNA (6 ng), and 48 μl ofwater. PCR began with a two minute incubation at 95° C., followed by 30cycles of 20 seconds at 95° C. for denaturation, 20 seconds forannealing at 57° C., and 20 seconds at 72° C. for extension. The samplewas incubated at 72° C. for three minutes and then held at 4° C. The PCRproduct was purified using a QIAquick® PCR Purification Kit (Qiagen,Valencia, Calif.). The bacterial expression vector pZS21-MCS and the Ec'tesA PCR product were digested with XhoI and HindIII. The digestedvector and insert were ligated using Fast-Link (EpicentreBiotechnologies, Madison, Wis.). The ligation mix was then used totransform E. coli TOP10 cells (Invitrogen, Carlsbad, Calif.).Recombinant plasmids were isolated using a QIAPrep® Spin Miniprep Kit(Qiagen) and characterized by gel electrophoresis of restriction digestswith HaeII. DNA sequencing confirmed that the 'tesA insert had beencloned and that the insert encoded the expected amino acid sequence (SEQID NO: 45). The resulting plasmid was designated pZS22 Ec 'tesA.

An E. coli strain deficient in fatty acid degradation (Voelker, T. A.,and H. M. Davies. J. Bacteriology 176: 7320-7 (1994)) and able toregulate transcription of recombinant genes was generated as follows. AnE. coli K-12 strain (K27) defective in fadD lacks the fatty acyl-CoAsynthetase responsible for an initial step in fatty acid degradation.The strain K27 (F—, tyrT58(AS), fadD88, mel-1; CGSC Strain #5478) wasobtained from the E. coli Genetic Stock Center (New Haven, Conn.). Agenomic regulation cassette from strain DH5αZ1 [lacl^(q), PN25-tetR,Sp^(R), deoR, supE44, Δ(lacZYA-argFV169), φ80 lacZΔM15 (Expressys,Ruelzheim, Germany)] was introduced into the host strain. Thetransducing phage P1vir was charged with DH5αZ1 DNA as follows. Alogarithmically growing culture (5 ml LB broth containing 0.2% glucoseand 5 mM CaCl₂) of donor strain, DH5αZ1, was infected with a 100 μl of alysate stock of P1vir phage. The culture was further incubated threehours for the infected cells to lyse. The debris was pelleted, and thesupernatant was further cleared through a 0.45 μm syringe filter unit.The fresh lysate was titered by spotting 10 μl of serial 1:10 dilutionsof lysate in TM buffer (10 mM MgSO₄/10 mM Tris.Cl, pH 7.4) onto a 100 mmLB (with 2.5 mM CaCl₂) plate overlayed with a cultured lawn of E. coliin LB top agar (with 2.5 mM CaCl₂). The process was repeated using thenewly created phage stock until the phage titer surpassed 10⁹ pfu/ml.

The higher titer phage stock was used to transduce fragments of theDH5αZ1 genome into a recipient K27 strain. An overnight culture (1.5 ml)of K27 was pelleted and resuspended in 750 μl of a P1 salts solution (10mM CaCl₂/5 mM MgSO₄). 100 μl of the suspended cells was inoculated withvarying amounts of DH5αZ1 donor P1vir lysate (1, 10, and 100 μl) insterile test tubes. The phage was allowed to adsorb to the cells for 30minutes at 37° C. Absorption was terminated by addition of 1 ml LB brothplus 200 μl of 1 M sodium citrate, and the cultures were furtherincubated for 1 hour at 37° C. with aeration. The cultures werepelleted, and the cells suspended in 100 μl of LB broth (plus 0.2 Msodium citrate) and spread onto LB agar plates with 50 μg/mLspectinomycin. Spectinomycin-resistant strains were isolated, andgenomic DNAs were screened by PCR for the presence of tetR, lacI^(q) andfadD88. One such transductant was named K27-Z1 and used in furtherstudies.

To transform K27-Z1, competent cells were placed on ice in pre-chilled14 ml round bottom centrifuge tubes. Each plasmid was incubated with 50μl of chemically competent K27-Z1 cells (Cohen, S. N., Change, A. C. Y.,and L. Hsu. Proceedings National Academy Sciences U.S.A. 69: 2110-4(1972)) for 30 minutes. The cells were heat shocked at 42° C. for 90seconds and immediately placed on ice for two minutes. Pre-warmed SOCmedium (250 μl) (Invitrogen, Carlsbad, Calif.) was added, and the cellswere allowed to recover at 37° C. with 125 rpm shaking for one hour.Transformed cell mix (20 μl) was spread onto selective LB agar with 100μg/ml ampicillin to select for cells carrying the pTrcHisA-basedplasmids. Transformed cell mix (50 μl) was spread onto LB agar with 34μg/ml chloramphenicol to select for cells carrying the pZA31-basedplasmids. Transformed cell mix (150 μl) was spread onto LB agar with 100μg/ml ampicillin and 34 μg/ml chloramphenicol to select for cellscarrying both the pTrcHisA-based and pZA31-based plasmids. In somecases, the creation of triple transformants required twotransformations: a double transformant was originally created, madecompetent, and transformed by a third plasmid.

Using the methods described above, E. coli strain K27-Z1 was transducedwith pTrcHisA pZA31 (control), pZA31 mutAB Ss epi, pTrcHisA Ec sbm, andpTrcHisA Ec sbm/pZA31 Mb mmat. The bacteria were cultured in M9 withglycerol (0.2%) at 22° C. in flasks that were coated with black Scotchduct tape. After the bacteria reached an optical density (600 nm) of0.4, a mix of IPTG, anhydrotetracycline, arabinose and hydroxocobalaminhydrochloride was added to the culture, giving final concentrations of 1mM, 100 ng/ml, 0.2%, and 20 μM, respectively. Twenty-four hours later,the bacteria were harvested for coenzyme A analysis. Methylmalonyl-CoAproduction is illustrated in FIG. 24. Host cells producing exogenousmethylmalonyl-CoA mutase and methylmalonyl-CoA epimerase (encoded bypZA31 mutAB Ss epi) produced over 25 ng methylmalonyl-CoA per mlculture. Host cells comprising additional copies of the Sbm(methylmalonyl-CoA mutase) coding sequence produced over three times theamount of methylmalonyl-CoA per ml of culture, and co-expression of anmethylmalonyl-CoA acyl transferase reduced the amount ofmethylmalonyl-CoA present in the culture medium.

Production of methylmalonyl-CoA in host cells expressing exogenouspropionyl-CoA carboxylase also was studied and is illustrated in FIG.25. BW25113 (control) and BW25113 containing pZA31-accA1-pccB (labeledas Pcc in the figure) were cultured in LB, and the coenzyme-A thioesterswere isolated and characterized as described above. Host cellscomprising a polynucleotide encoding an exogenous propionyl-CoAcarboxylase produced over about 15 ng methylmalonyl-CoA per ml ofculture.

When Ec 'tesA was present, less longer-chain (fifteen and seventeencarbons) and more mid-chain (thirteen carbons) branched fatty acids wereproduced by the host cell, indicating that production of thioesteraseincreases the proportion of medium chain-length branched fatty acidsproduced by the inventive method.

Example 16 Analysis of Scattered Branched Fatty Acid by Two-Dimensional(2D) Gas Chromatography

To identify branched fatty acids produced by recombinant E. coliproduced as described herein, fatty acids were isolated from bacterialcultures and derivatives were generated to facilitate identification.The fatty acid derivatives were separated by 2D gas chromatography andmass spectrometry was used to characterize fragmented samples.Derivatization of fatty acids to their 4,4′ dimethyloxazolinederivatives prior to analysis via mass spectrometry has been described(Zhang, J. Y., QT. Yu, B. N. Liu and Z. H. Huang, Biomed Env. MassSpectrom. 15:33 (1988)). By careful examination of minor spectraldifferences, it possible to determine the location of branch points onthe backbones of fatty acid derivatives.

One liter of bacterial samples in LB (modified to contain only 0.5 mg/mlsodium chloride, unless otherwise indicated) with cyanocobalamin (20 μM)were cultured at 22° C. for 25 hours following induction with IPTG,anhydrotetracycline, and arabinose. A cell pellet was collected bycentrifugation at 3500 rpm, and the supernatant was discarded. The cellpellet was suspended in the remaining liquid, and the slurry wastransferred into Pyrex tubes (#9826, Corning Inc., Lowell, Mass.). Anequal volume of chloroform was added, and the sample was dried at roomtemperature overnight.

To produce samples for analysis, cell pellets (0.5 grams) were placed ina round bottom flask, and 0.5 grams of KOH pellets and 25 ml of waterwere added. The E. coli pellets and KOH solution were refluxed for threehours, and the sample was allowed to cool. Concentrated HCl was addeddrop-wise, using a methyl orange endpoint to ensure fatty carboxylicacids were in the acid form. The acidified aqueous solution was thenextracted three times with 25 ml aliquots of hexane to extract the fattyacids into the organic layer.

To convert fatty acid to oxazoline derivatives, the hexane extract wasevaporated to dryness and reconstituted into 5 ml of hexane to whichsodium sulfate was added as a drying agent. After evaporating the sampleto a 1 ml volume, a portion (0.6 ml) was decanted into a Reactitherm™vial. The hexane in the Reactitherm™ vial was again evaporated todryness, and 2 ml of 2-methyl-2-aminopropanol was added. The vial wascapped and heated for 4 hours at 200° C. The cooled2-methyl-2-aminopropanol solution was transferred to a scintillationvial, to which 5 ml of methylene chloride was added. The sample waswashed with three 5 ml volumes of water. Sodium sulfate was added to themethylene chloride to remove any residual water, and an aliquot wastransferred to a GC vial for analysis.

The derivatized samples were analyzed on a Leco Pegasus 4D Comprehensive2D gas chromatograph time-of-flight mass spectrometer equipped with a30M Supelco GammaDex 120 (Supelco 24307) column in the first dimensionand a 2M Varian VF5-MS (Varian CP9034) column in the second dimension.Retention times of key chain-length fatty acids (in both first andsecond dimensions) in test samples were confirmed by identicalpreparation and analysis of a Supleco (47080-U) BAME (bacterial acidmethyl ester) standard mixture. Using these columns, 4,4′dimethyloxazoline-derivatized branched-chain fatty acids were expectedto elute prior to their linear chain-length homologs in the firstdimension, and this was confirmed by the iso and anteiso structuralisomers of C15 methyl esters (derivatized to their4,4′-dimethyloxazoline derivatives) in the BAME standard referenceabove.

The profile of fatty acids produced by two strains was compared. Thefirst strain was engineered to produce branched fatty acids [BL21 Star(DE3) (pTrcHisA Ec sbm So ce epi pZA31 mmat)] and the second was acontrol strain [BL21 Star (DE3) (pTrcHisA pZA31)]. A sample of E. coliBL21 Star (DE3) comprising pTrcHisA Ec sbm So ce epi and pZA31 mmat wasdeposited with American Type Culture Collection (ATCC), 10801 UniversityBlvd., Manassas, Va., on Dec. 14, 2010, under the provisions of theBudapest Treaty for the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure (“Budapest Treaty”),and assigned Deposit Accession No. [XXX] on [DATE]. The sample from thefirst strain revealed several peaks in the region where branched fattyacids were expected (FIG. 26), whereas the sample from the controlstrain revealed no such peaks (FIG. 27). For example, several peaks(labeled 54, 55, and 57) were in a position consistent with branched C15acids, and peaks 137 and 139 were in a position expected for branchedC17 acids. Mass spectrometry established that these peaks comprisebranched fatty acids.

The mass spectral fragmentation pattern of oxazoline derivatives wasused to confirm that the fatty acids identified using 2D GC containedbranches. Oxazoline derivatives fragment along the length of the carbonchain starting from the functional end of the molecule. If a branchpoint occurs along the backbone, there is a gap in the mass spectrumpattern; which peak is missing (or reduced) depends on the location ofthe branch. FIG. 28 depicts the mass spectra of the peaks labeled 54,55, and 57 in FIG. 26 as oxazoline derivatives of methyl-branchedtetradecanoic fatty acids. The ions circled exhibit reduced or nointensity relative to the reference spectrum of linear pentadecanoicfatty acid (bottom spectrum), and were assigned as 8-methyl, 10-methyl,and 12-methyl (anteiso) tetradecanoic fatty acid (all as oxazolinederivatives). Peak 57 was tentatively identified as the anteiso C15oxazoline derivative despite the similarity to the mass spec data forthe linear sample because 1) peak 61 migrated at the position of ananteiso C15 standard on 2D gas chromatography, 2) the 252 molecularweight ion is present in slightly lower amounts relative to the nearby238 and 266 molecular weight ions, and 3) anteiso compounds can bedifficult to identify by this technique. The 8- and 10-branched fattyacids are shown in the top two profiles of FIG. 28, readily identifiedby the almost complete absence of the fragment circled. Peaks 137 and139 in FIG. 26 were assigned as 8-methylhexadecanoic acid and12-methylhexadecanoic acids (as oxazoline derivatives). Thus, B132 Star(DE3) (pTrcHisA Ec sbm So ce epi pZA31 mmat) (i.e., a recombinantmicrobe comprising overexpressed or recombinant polynucleotides encodinga methylmalonyl-CoA mutase, a methylmalonyl-CoA epimerase, and an acyltransferase) generated branched-chain C15 and C17 fatty acids comprisingmethyl branches on even-number carbons.

Branched fatty acid production also was observed in host cells producingexogenous propionyl-CoA carboxylase and Streptomyces coelicolormethylmalonyl-CoA mutase. The propionyl-CoA carboxylase gene-containingstrain produced the branched fatty acids shown in Table F.

TABLE F Molecular Weight as fatty Peak # Proposed Compound ID FormulaDMOX acid 38 6-methyl, dodecanoic acid C₁₃H₃₃ 267 214 (DMOX) (C₄H₈NO) 408-methyl, dodecanoic acid C₁₃H₃₃ 267 214 (DMOX) (C₄H₈NO) 61 6-methyl,tridecanoic acid C₁₄H₃₅ 281 228 (DMOX) (C₄H₈NO) 62 8-methyl, tridecanoicacid C₁₄H₃₅ 281 228 (DMOX) (C₄H₈NO) 101 6-methyl, tetradecanoic acidC₁₅H₃₇ 295 242 (DMOX) (C₄H₈NO) 103 10-methyl, tetradecanoic acid C₁₅H₃₇295 242 (DMOX) (C₄H₈NO) 140 10-methyl, pentadecanoic acid C₁₆H₃₉ 309 256(DMOX) (C₄H₈NO) 182 8-methyl, hexadecanoic acid C₁₇H₄₁ 323 270 (DMOX)(C₄H₈NO) 189 12-methyl, hexadecanoic acid C₁₇H₄₁ 323 270 (DMOX) (C₄H₈NO)

The S. coelicolor methylmalonyl-CoA mutase gene-containing microbe (BL21Star (DE3) harboring pZA31 mutAB Ss epi pTrcHisA mmat) produced fourbranched fatty acids: 6-methyltetradecanoic acid, 10-methyltetradecanoicacid, 6-methylhexadecanoic acid, and 12-methylhexadecanoic acid.

Using 2D gas chromatography and mass spectrometry, fatty acid profileswere compared for two recombinant strains comprising Ec sbm, So ce epi,Mb mmat and containing or lacking a thioesterase coding sequence('tesA). The amount of branched C15 fatty acids relative to branched C17fatty acids was greater in the 'tesA-containing strain. The area percentratio of branched C15 fatty acid to branched C17 fatty acids in K27-Z1(pTrcHisA Ec sbm So ce epi pZA31 mmat) was 1.4, while the ratio producedby K27-Z1 (pTrcHisA Ec sbm So ce epi pZA31 mmat pZS22 Ec 'tesA) was 7.0.Expression of a thioesterase shortened the chain length of branchedfatty acids.

These results demonstrate that a cell of the invention producingpropionyl-CoA carboxylase or producing methylmalonyl-CoA mutase,methylmalonyl-CoA epimerase, and acyl transferase generatesbranched-chain fatty acids comprising methyl branches on even-numbercarbons. Recombinant host cells further comprising a polynucleotideencoding a thioesterase preferentially produce fatty acid comprisingshorter chain length.

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “40 mm” is intended to mean“about 40 mm.”

Every document cited herein, including any cross referenced or relatedpatent or application, is hereby incorporated herein by reference in itsentirety unless expressly excluded or otherwise limited. The citation ofany document is not an admission that it is prior art with respect toany invention disclosed or claimed herein or that it alone, or in anycombination with any other reference or references, teaches, suggests ordiscloses any such invention. Further, to the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

While particular embodiments of the invention have been illustrated anddescribed, it would be obvious to those skilled in the art that variousother changes and modifications can be made without departing from thespirit and scope of the invention. It is therefore intended to cover inthe appended claims all such changes and modifications that are withinthe scope of this invention.

1. A method for producing branched-chain fatty acid comprising a methylon one or more even number carbons, the method comprising culturing acell comprising (aa) an exogenous or overexpressed polynucleotidecomprising a nucleic acid sequence encoding a polypeptide that catalyzesthe conversion of propionyl-CoA to methylmalonyl-CoA and/or (bb) anexogenous or overexpressed polynucleotide comprising a nucleic acidsequence encoding a polypeptide that catalyzes the conversion ofsuccinyl-CoA to methylmalonyl-CoA, under conditions allowing expressionof the polynucleotide(s) and production of branched-chain fatty acid,wherein the cell produces more branched-chain fatty acid comprising amethyl on one or more even number carbons than an otherwise similar cellthat does not comprise the polynucleotide(s).
 2. The method of claim 1further comprising extracting from culture the branched-chain fatty acidor a product of the branched-chain fatty acid.
 3. The method of claim 2,wherein the polypeptide that catalyzes the conversion of propionyl-CoAto methylmalonyl-CoA is a propionyl-CoA carboxylase and/or thepolypeptide that catalyzes the conversion of succinyl-CoA tomethylmalonyl-CoA is a methylmalonyl-CoA mutase.
 4. The method of claim3, wherein (i) the propionyl-CoA carboxylase is Streptomyces coelicolorPccB and AccA1 or PccB and AccA2 and/or (ii) the methylmalonyl-CoAmutase is Janibacter sp. HTCC2649 methylmalonyl-CoA mutase, S.cinnamonensis MutA and MutB, or E. coli Sbm.
 5. The method of claim 3,wherein (i) the methylmalonyl-CoA mutase comprises an amino acidsequence having at least about 80% sequence identity to the amino acidsequence set forth in SEQ ID NOs: 3, 4, or 28 and/or (ii) thepropionyl-CoA carboxylase comprises an amino acid sequence having atleast about 80% sequence identity to the amino acid sequence set forthin SEQ ID NOs: 9 and
 10. 6. The method of claim 3, wherein the cellcomprises an exogenous or overexpressed polynucleotide comprising anucleic acid sequence encoding a methylmalonyl-CoA mutase and furthercomprises an exogenous or overexpressed polynucleotide comprising anucleic acid sequence encoding a methylmalonyl-CoA epimerase.
 7. Themethod of claim 2, wherein the cell further comprises an exogenous oroverexpressed polynucleotide encoding an acyl transferase lackingpolyketide synthesis activity and/or an exogenous or overexpressedpolynucleotide comprising a nucleic acid sequence encoding athioesterase.
 8. The method of claim 7, wherein the acyl transferase isFabD, an acyl transferase domain of a polyketide synthase, or an acyltransferase domain of Mycobacterium mycocerosic acid synthase.
 9. Themethod of claim 2, wherein the cell has been modified to attenuateendogenous methylmalonyl-CoA mutase activity, endogenousmethylmalonyl-CoA decarboxylase activity, and/or endogenous acyltransferase activity.
 10. The method of claim 2, wherein the cellproduces a Type II fatty acid synthase.
 11. The method of claim 10,wherein the cell is Escherichia coli.
 12. A branched-chain fatty acidproduced by the method of claim
 1. 13. A cell comprising: (i) anexogenous or overexpressed polynucleotide comprising a nucleic acidsequence encoding an acyl transferase lacking polyketide synthesisactivity, and (ii) an exogenous or overexpressed polynucleotidecomprising a nucleic acid sequence encoding a propionyl-CoA carboxylaseand/or an exogenous or overexpressed polynucleotide comprising a nucleicacid sequence encoding a methylmalonyl-CoA mutase, wherein thepolynucleotide(s) are expressed and the cell produces morebranched-chain fatty acid comprising a methyl on one or more even numbercarbons than an otherwise similar cell that does not comprise thepolynucleotide(s).
 14. The cell of claim 13, wherein (i) thepropionyl-CoA carboxylase is Streptomyces coelicolor PccB and AccA1 orPccB and AccA2 and/or (ii) the methylmalonyl-CoA mutase is Janibactersp. HTCC2649 methylmalonyl-CoA mutase, S. cinnamonensis MutA and MutB,or E. coli Sbm.
 15. The cell of claim 13, wherein (i) themethylmalonyl-CoA mutase comprises an amino acid sequence having atleast about 80% sequence identity to the amino acid sequence set forthin SEQ ID NOs: 3, 4, or 28 and/or (ii) the propionyl-CoA carboxylasecomprises an amino acid sequence having at least about 80% sequenceidentity to the amino acid sequence set forth in SEQ ID NOs: 9 and 10.16. The cell of claim 13, wherein the cell comprises an exogenous oroverexpressed polynucleotide comprising a nucleic acid sequence encodinga methylmalonyl-CoA mutase and further comprises an exogenous oroverexpressed polynucleotide comprising a nucleic acid sequence encodinga methylmalonyl-CoA epimerase.
 17. The cell of claim 13, wherein theacyl transferase is FabD, an acyl transferase domain of a polyketidesynthase, or an acyl transferase domain of Mycobacterium mycocerosicacid synthase.
 18. The cell of claim 13, wherein the cell furthercomprises an exogenous or overexpressed polynucleotide comprises anucleic acid sequence encoding a thioesterase.
 19. The cell of claim 13,wherein the cell has been modified to attenuate endogenousmethylmalonyl-CoA mutase activity, endogenous methylmalonyl-CoAdecarboxylase activity, and/or endogenous acyl transferase activity. 20.The cell of claim 13, wherein the cell is Escherichia coli.